Direct differentiation of human pluripotent stem cells and characterization of differentiated cells

ABSTRACT

This invention provides a system for efficiently producing differentiated cells from pluripotent cells, such as human embryonic stem cells. Rather than permitting the cells to form embryoid bodies according to established techniques, differentiation is effected directly in monolayer culture on a suitable solid surface. The cells are either plated directly onto a differentiation-promoting surface, or grown initially on the solid surface in the absence of feeder cells and then exchanged into a medium that assists in the differentiation process. The solid surface and the culture medium can be chosen to direct differentiation down a particular pathway, generating a cell population that is remarkably uniform. The methodology is well adapted to bulk production of committed precursor and terminally differentiated cells for use in drug screening or regenerative medicine.

RELATED APPLICATIONS

[0001] This application claims priority to the following U.S.provisional patent applications: U.S. Ser. No. 60/213,739, filed Jun.22, 2000; U.S. Ser. No. 60/216,387, filed Jul. 7, 2000; and U.S. Ser.No. 60/220,064, filed Jul. 21, 2000.

[0002] The aforelisted priority applications are hereby incorporatedherein by reference in their entirety, as are each of the following:U.S. Ser. No. 60/175,581, filed Jan. 11, 2000; U.S. Ser. No. 09/688,031,filed Oct. 10, 2000; U.S. Ser. No. 09/718,308, filed Nov. 20, 2000, U.S.Ser. No. 60/257,608, filed Dec. 12, 2000; International PatentApplication PCT/US01/01030, filed Jan. 10, 2001; International PatentApplication PCT/US01/13471, filed Apr. 26, 2001; and U.S. Ser. No.09/859,351, filed May 16, 2001.

TECHNICAL FIELD

[0003] This invention relates generally to the field of cell biology ofembryonic cells. More specifically, it relates to conditions that allowhuman pluripotent stem cells to be directly differentiated into cells ofa particular lineage, suitable for applications such as use in tissueregeneration and the screening of biologically active substances.

BACKGROUND

[0004] Recent discoveries have raised expectations that stem cells maybe a source of replacement cells and tissues that are damaged in thecourse of disease, infection, or because of congenital abnormalities.Various types of putative stem cells differentiate when they divide,maturing into cells that can carry out the unique functions ofparticular tissues, such as the heart, the liver, or the brain.

[0005] A particularly important discovery has been the development ofpluripotent stem cells, which are thought to have the potential todifferentiate into almost any cell type. The next challenge indeveloping the technology is to obtain dependable conditions for drivingdifferentiation towards particular cell lineages that are desired fortherapeutic purposes.

[0006] Early work on embryonic stem cells was done in mice (reviewed inRobertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil.Dev. 6:543, 1994). Most methods of differentiating mouse pluripotentstem cells involve three strategies, often in combination:

[0007] Permitting the cells to form aggregates or embryoid bodies, inwhich cells interact and begin to differentiate into a heterogeneouscell population with characteristics of endoderm, mesoderm, and ectodermcells. The embryoid bodies are then harvested and cultured further sothat the differentiation can continue.

[0008] Inducing the cells to differentiate using soluble factors thatpromote particular forms of differentiation, optionally withsimultaneous withdrawal of factors that inhibit differentiation

[0009] Transfecting the cells with a tissue-specific gene, that has theeffect of directing the cell towards the tissue type desired

[0010] Mummery et al. (Cell Differentiation Dev. 30:195, 1990) comparedcharacteristics of mouse embryonic stem (ES) cells with two embryonalcarcinoma lines. The cells were differentiated either by letting cellsform aggregates, optionally in the presence of retinoic acid (RA) ordimethyl sulfoxide (DMSO); or letting the cells grow to confluence,optionally depriving the culture of leukemia inhibiting factor (LIF) ordifferentiation inhibiting activity (DIA) found in high concentrationsin medium conditioned by Buffalo rat liver (BRL) cells. The studysuggested that mixed endoderm-mesoderm cells were obtained afterremoving inhibitors of differentiation, and parietal endoderm-like cellswere obtained by RA induction.

[0011] Grendon et al. (Dev. Biol. 177:332, 1996) generated anendothelial cell line capable of embryonic vasculogenesis from mouse EScells. The cells were transfected with the early region of SV40 Large Tantigen, and then cultured in medium comprising homogenized mousetestes, which promotes differentiation. An endothelial line was derivedthat expresses endothelial cell specific proteins and can be induced bybasic fibroblast growth factor (bFGF) and LIF to proliferate to formvascular tubes and microcapillary anastomoses.

[0012] Van Inzen et al. (Biochim. Biophys. Acta 1312:21, 1996)differentiated mouse embryonic stem cells by incubating the cells for atleast 3 days with retinoic acid. The cells were cultured either as amonolayer, or as embryoid bodies on a non-adhesive substrate. The cellsobtained from culture stained positively for the neuronal markers NF-165and GAP-43, and were electrically excitable in a patch clamp assay.

[0013] Dinsmore et al. (Cell Transplant. 5:131, 1996) report a methodfor controlled differentiation of mouse embryonic stem cells in vitro toproduce populations containing neurons or skeletal muscle cells.Embryoid bodies were allowed to form, and were induced using dimethylsulfoxide (DMSO) to differentiate to muscle cells, or using retinoicacid to differentiate to neurons. Muscle cells were also made bytransfecting ES cells with an expression vector for muscle-specificprotein MyoD.

[0014] Rathjen et al. (J. Cell Sci. 112:601, 1999, and InternationalPatent Publication WO 99/53021) formed a primitive ectoderm-like (EPL)cell population from mouse ES cells using conditioned medium from thehuman hepatocarcinoma line HepG2. When grown in medium without feedercells, but including LIF, the mouse ES cells reportedly grew as ahomogeneous population with most colonies displaying domed morphology.Differentiation was effected by culturing the mouse ES cells in thepresence of LIF and HepG2 conditioned medium. This gave rise to amorphologically distinct population of EPL cells with differentphenotypic markers and altered differentiation properties. EPL cellformation was reversible in the presence of LIF by withdrawing theconditioned medium.

[0015] Tropepe et al. (Soc. Neuroscience 25: abstract 205.18, 1999)reported that a small percentage of mouse ES cells proliferate inserum-free low-density conditions in the presence of LIF, and formsphere colonies that may subsequently differentiate into neurons andglia. A small proportion of cells from primary colonies can generatesecondary colonies independent of LIF but dependent on the factor FGF2.Blocking BMP signaling by adding noggin protein increases the proportionof cells forming neural stem cells. About 60% of single ES cellscultured for 24 h in serum-free medium express nestin.

[0016] Pluripotent stem cells of human origin

[0017] Work on human pluripotent stem (hPS) cells has been more than adecade behind the experiments conducted on mouse cells. Human PS cellsare more fragile and more difficult to isolate. Furthermore, they cannotbe maintained in an undifferentiated state under conditions developedfor mouse cells.

[0018] Recently, some of these challenges have been overcome. Thomson etal. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995)were the first to successfully culture stem cells from non-humanprimates. Thomson et al. also derived human embryonic stem (hES) celllines from human blastocysts (Science 282:114, 1998). Gearhart andcoworkers derived human embryonic germ (hEG) cell lines from fetalgonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726,1998 and International Patent Publication No. WO 98/43679). Both hES andhEG cells have the long-sought characteristics of hPS cells: they arecapable of long-term proliferation in vitro without differentiating,they retain a normal karyotype, and they retain the capacity todifferentiate to a number of different derivatives.

[0019] Human pluripotent stem cells differ from mouse ES cells in anumber of important respects. Thomson et al. and Gearhart et al.maintained their hPS cells in an undifferentiated state by culturing ona layer of embryonic feeder cells. In contrast, mouse ES cells can begrown easily without feeder cells in appropriate conditions,particularly the presence of leukemia inhibiting factor (LIF) or otherligands that bind receptors that associate with gp130. However, LIFalone has not been reported to prevent differentiation of hPS in theabsence of feeders. Another difference is that mouse ES cells can beplated in a completely dispersed fashion; and grow quite happily toproduce undifferentiated ES progeny. In contrast, single hES cells areunstable; and propagation of hES cells typically requires that they bepassaged as clusters of cells during each replating.

[0020] Current efforts to differentiate hPS cells involve the formationof cell aggregates, either by overgrowth of hPS cells cultured onfeeders, or by forming embryoid bodies in suspension culture. Theembryoid bodies generate cell populations with a highly heterogeneousmixture of phenotypes, representing a spectrum of different celllineages—which depends in part on the size of each aggregate and theculture conditions.

[0021] Large-scale commercial production of committed precursor cells orfully differentiated cells from hPS cells would require adifferentiation protocol that did not involve producing cell aggregatesor embryoid bodies. In addition, there is a need for cell populationsthat have relatively uniform and reproducible characteristics for use indrug screening and human therapy.

[0022] Accordingly, there is a need for new technology that facilitatesderivation of differentiated cells from human pluripotent stem cells.

SUMMARY

[0023] This invention provides a system for efficient production ofdifferentiated cells from primate pluripotent stem (pPS) cells. Ratherthan permitting the pPS to form embryoid bodies, differentiation iseffected directly by plating sub-confluent cultures of pPS cells onto asolid surface that facilitates differentiation, in the absence of feedercells or culture conditions that simulate the presence of feeder cells.The nature of the solid surface and components of the culture medium canbe chosen to direct differentiation down a cell lineage pathway that isdesired for research or therapeutic use.

[0024] Embodied in this invention are methods for directly obtainingdifferentiated cells from a donor culture of undifferentiated pPS cells,without forming embryoid bodies. Undifferentiated cells are newly platedonto a solid surface, or otherwise exchanged into a new cultureenvironment that induces differentiation of the cells into the desiredphenotype in a direct fashion, without overgrowth, aggregate formation,or otherwise creating the condensed heterogeneous cell populationcharacteristic of embryoid bodies.

[0025] One way of accomplishing this is to prepare a suspension of cellsfrom an undifferentiated donor culture; replate and culture thesuspended cells on a solid surface so that they differentiate withoutforming embryoid bodies; and harvest differentiated cells from the solidsurface. A variation is to harvest pPS cells from the donor culturebefore there is overgrowth or formation of colonies; replate and culturethe harvested cells on a solid surface so that they differentiate; andharvest differentiated cells from the solid surface. Another variationis to prepare a suspension from a culture of both pPS cells and feedercells; replate and culture the suspended cells on a solid surfacewithout adding fresh feeder cells; and harvest differentiated cells fromthe solid surface. A further variation is to provide a donor culturecomprising undifferentiated pPS cells growing on an extracellular matrixin the absence of feeder cells; prepare a suspension of cells from thedonor culture; replate and culture the suspended cells on a solidsurface without the extracellular matrix; and harvest differentiatedcells from the solid surface.

[0026] A further variation is to provide a culture of primatepluripotent stem (pPS) cells that is essentially free of feeder cells;change the medium in which the cells are cultured; and harvestdifferentiated cells from the culture after culture for a sufficientperiod to effect differentiation in the changed medium. The medium maybe changed either by replacing the medium in the culture with a freshmedium having a new composition, or by adding new constituents to themedium already present and then continuing the culture.

[0027] In any of these embodiments, the pPS cells are any type of cellcapable of forming progeny of each of the three germ layers, exemplifiedbut not limited to human embryonic stem (hES) cells. The replating canbe performed without selecting a particular cell population from thesuspended (or harvested) cells. For example, the replated cellsuspension can be obtained by incubating the donor culture withcollagenase or trypsin or E.D.T.A., thereby releasing the cells from asurface to which the cells adhere, and collecting the released cells ina suitable medium. The replating can be done in the absence of freshlyadded feeder cells or extracellular matrix proteins on the solidsurface, such as a glass cover slip, optionally bearing a polycationsuch as polyornithine or polylysine.

[0028] Differentiation can be promoted by withdrawing serum or serumreplacement from medium, withdrawing a factor that promotesproliferation, withdrawing a factor that inhibits differentiation, oradding a new factor that promotes differentiation. Exemplary factors forgenerating neuronal cells are Brain Derived Neurotrophic Factor (BDNF)and Neutrotrophin-3 (NT-3).

[0029] A proportion of the cells cultured according to this inventionmay differentiate to precursor cells committed to a restricted celllineage and capable of proliferation, such as ectodermal cells (forexample, neuroectoderm lineage), mesodermal cells, or cells of theendoderm or visceral endoderm. A proportion of the cells may becomefully differentiated cells, such as neurons or glial cells. If desired,the harvested committed precursor cells or fully differentiated cellscan optionally be genetically altered with a polynucleotide that encodestelomerase.

[0030] The differentiated cells of this invention may be used to screencandidate compounds or environmental conditions that affectdifferentiation or metabolism of a cell type of interest. Thedifferentiated cells may be used to obtain cell specific antibodypreparations and cell-specific cDNA libraries, to study patterns of geneexpression, or as an active ingredient in a pharmaceutical preparation.

[0031] The differentiated cells of this invention can also be used toidentify a substance expressed at a different level in committed ordifferentiated cells compared with undifferentiated primate pluripotentstem (pPS) cells. Such substances may include but are not limited tomRNA transcripts, secreted protein, intracellular protein, cell-surfaceprotein, cell-surface oligosaccharide, and particular lipids organgliosides. Expression may be compared, for example, at the level oftranscription, translation, surface presentation, or enzymatic activity.Expression of oligosaccharide and lipid substances can be inferred bychemical or antibody analysis, or by deduction from expression ofenzymes required for their synthesis. Particular embodiments involvedetermining the level of expression of a plurality of mRNAs in committedor differentiated cells made by direct differentiation, embryoid bodyformation, or any other suitable technique, and comparing the leveldetermined with the level of expression of the same mRNAs in anothercell type, such as undifferentiated pPS cells. A polynucleotide can thenbe prepared that shares sequence with mRNA that is expressed at adifferent level in the differentiated cells.

[0032] These and other embodiments of the invention will be apparentfrom the description that follows.

DRAWINGS

[0033]FIG. 1 provides an analysis of OCT-4 and hTERT expression in hEScells cultured with feeder cells (mEF) or extracellular matrix(Matrigel® or laminin) with regular medium (RM) or conditioned medium(CM). The upper panel is a copy of a gel showing OCT-4 and hTERTexpression at the mRNA level by RT-PCR. The lower panel is a bar graphcomparing the level of expression for cells grown on differentsubstrates, expressed as the ratio of OCT-4 or hTERT to the 18sstandard. hES cells grown on Laminin and Matrigel® in conditioned mediumhave similar expression patterns to those of cells grown on a feederlayer.

[0034]FIG. 2 is a half-tone reproduction of a phase contrastphotomicrograph (10X, 40X), showing cells at various times during directdifferentiation to a hepatocyte phenotype. Row A shows cells 4 daysafter culture in SR medium containing 5 mM sodium n-butyrate. More than80% of cells in the culture are large in diameter, containing largenuclei and granular cytoplasm. After 5 days, the cells were switched tospecialized hepatocyte culture medium (HCM). Rows B and C show theappearance after culturing in HCM for 2 or 4 days. Multinucleatedpolygonal cells are common. By these criteria, the directlydifferentiated ES-derived cells resemble freshly isolated human adulthepatocytes (Row D) and fetal hepatocytes (Row E).

[0035]FIG. 3 is a matrix chart, representing relative expression of mRNAin embryoid body (EB) cells, compared with expression in theundifferentiated hES cell line from which they were derived. Probes usedto analyze expression are listed to the left. The first three columns ofthe matrix show the kinetics of relative expression for EB cellscultured for 2, 4, or 8 days. The fourth column (4d−/4d+) shows relativeexpression of EB cells to which retanoic acid was added for the final 4days of culture.

[0036]FIG. 4 is a reproduction of a fluorescence micrograph, showingneuronal cells obtained by direct differentiation of ES cells on a solidsubstrate using a mixture of differentiation factors. The three fieldsshown were all taken from treatments that comprised neurotrophins andthe TNF-β superfamily antagonists noggin and follistatin. A number ofcells are seen that have neuronal processes and stain for the neuronalmarker β-tubulin-III. The proportion of MAP-2 positive cells that werealso positive for tyrosine hydroxylase (a marker for dopaminergicneurons) was as high as ˜15%.

DETAILED DESCRIPTION

[0037] This invention provides a system for directly differentiatingprimate pluripotent stem (pPS) cells into committed precursor cells orfully differentiated cells. The system avoids forming aggregates orembryoid bodies as an intermediate step. hPS cells are maintained as amonolayer or dispersed from a sub-confluent pPS culture, and plated ontoa suitable substrate in an appropriate culture environment that promotesdifferentiation.

[0038] Before this invention was made, the expectation was that astrategy avoiding embryoid bodies would be unsuccessful. Classicaldevelopmental biology suggests that interaction between the threegerminal layers of the embryo is essential for appropriatedifferentiation. Embryoid bodies are reminiscent of this early stage ofdevelopment, in that they form the three germ layers in juxtaposition.

[0039] For example, nervous tissue is formed from the ectoderm. Recentevidence from the expression patterns of a number of genes suggests thatanother germ layer—the primitive endoderm—is involved in specifyingneural fate in the mouse (Bouwmeester et al., BioEssays 19:855, 1997;reviewed in Davidson et al., “Cell lineage and Fate Determination”,Academic Press, 1999; pp. 491-504 at 498). Endoderm ablation experimentsstrongly implicate an interaction of two germ layers that successivelyexpress the Hesxl gene during gestation—which raises the possibilitythat such interactions may be critical for the specification of neuralfate of the epiblast. Chimeric embryos deficient in the nodal gene failto develop forebrain (Varlet et al., Development 124:1033, 1997).Results of nodal and hex1 studies strongly suggest that there is acritical requirement for the specification of neural cells in theepiblast. In addition, isolated epiblast from early embryos is unable toexpress brain specific genes Otx2, En1, and En2 unless they are culturedtogether with fragments of the mesoendoderm. This evidence suggests thatcells from all three germ layers participate in neural differentiationin the epiblast.

[0040] Cells from all three germ layers are present in embryoid bodies,and provide an amalgam of signaling that may be similar to signalingthat occurs during normal embryo development. But this type ofinteraction between different cell types is lacking when pPS cells areplated in monolayers.

[0041] Contrary to previous expectations, it has now been discoveredthat plating undifferentiated hES cells directly onto a suitable surfaceor changing medium in monolayer culture provides a system whereby adifferentiated cell population may be reliably derived—in spite of thesignaling one might expect to be lacking from cells of other germlayers.

[0042] In an exemplary experiment, cultures of rhesus and human ES linesgrown on feeder cells were harvested using collagenase, and the cellswere then dissociated to clusters of ˜50-100 cells. The cells were thenplated onto glass coverslips coated with poly-ornithine, and culturedfor 1 week. Cultures showed positive immunoreactivity for β-tubulin IIIIand MAP-2, markers that are characteristic of neurons; glial fibrillaryacidic protein (GFAP), which is characteristic of astrocytes; and GaIC,which is characteristic of oligodendrocytes—indicating that all threemajor cell phenotypes of the central nervous system were present.

[0043] Under optimized conditions, this system can provide a remarkablyconsistent population of differentiated cells, with less heterogeneitythan what is present in a population of embryoid-body derived cells.Example 5 of this disclosure illustrates that the direct differentiationmethod can be used to obtain populations that are highly enriched fordopaminergic neurons. The direct differentiation method provides animportant source of reproducible high-quality cells for use in therapyand drug screening.

[0044] Definitions

[0045] Prototype “primate Pluripotent Stem cells” (pPS cells) arepluripotent cells derived from pre-embryonic, embryonic, or fetal tissueat any time after fertilization, and have the characteristic of beingcapable under the right conditions of producing progeny of severaldifferent cell types. pPS cells are capable of producing progeny thatare derivatives of each of the three germinal layers: endoderm,mesoderm, and ectoderm, according to a standard art-accepted test, suchas the ability to form a teratoma in a suitable host.

[0046] Included in the definition of pPS cells are embryonic cells ofvarious types, exemplified by human embryonic stem (hES) cells, asdescribed by Thomson et al. (Science 282:1145, 1998); embryonic stemcells from other primates, such as Rhesus or marmoset stem cellsdescribed by Thomson et al. (Proc. NatI. Acad. Sci. USA 92:7844, 1995;Developmental Biology 38:133, 1998); and human embryonic germ (hEG)cells, described in Shamblott et al. (Proc. Natl. Acad. Sci. USA95:13726, 1998). Other types of pluripotent cells are also included inthe term. Any cells of primate origin that are capable of producingprogeny that are derivatives of all three germinal layers are included,regardless of whether they were derived from embryonic tissue, fetaltissue, or other sources. For many embodiments of the invention, it isbeneficial to use pPS cells that are karyotypically normal and notderived from a malignant source.

[0047] pPS cell cultures are described as “undifferentiated” or“substantially undifferentiated” when a substantial proportion of stemcells and their derivatives in the population display morphologicalcharacteristics of undifferentiated cells, clearly distinguishing themfrom differentiated cells of embryo or adult origin. UndifferentiatedpPS cells are easily recognized by those skilled in the art, andtypically appear in the two dimensions of a microscopic view with highnuclear/cytoplasmic ratios and prominent nucleoli. It is understood thatcolonies of undifferentiated cells within the population will often besurrounded by neighboring cells that are differentiated. Nevertheless,the undifferentiated colonies persist when the population is cultured orpassaged under appropriate conditions, and individual undifferentiatedcells constitute a substantial proportion of the cell population.Cultures that are substantially undifferentiated contain at least 20%undifferentiated pPS cells, and may contain at least 40%, 60%, or 80% inorder of increasing preference (in terms percentage of cells with thesame genotype that are undifferentiated). Using the methods described inthis disclosure, it is sometimes possible to develop or passage culturesthat contain a relatively low proportion of differentiated pPS cells(even as low as 5 or 10%) into cultures that are substantiallyundifferentiated.

[0048] Whenever a culture or cell population is referred to in thisdisclosure as proliferating “without differentiation”, what is meant isthat after proliferation, the composition is substantiallyundifferentiated according to the preceding definition. Populations thatproliferate through at least four passages (˜20 doublings) withoutdifferentiation will contain substantially the same proportion ofundifferentiated cells (or possibly a higher proportion ofundifferentiated cells) when evaluated at the same degree of confluenceas the originating culture.

[0049] “Feeder cells” or “feeders” are cells of one type that areco-cultured with cells of another type, to provide an environment inwhich the cells of the second type can grow. The feeder cells areoptionally from a different species as the cells they are supporting.For example, certain types of pPS cells can be supported by primarycultures of mouse embryonic fibroblasts, immortalized mouse embryonicfibroblasts, or human fibroblast-like cells differentiated from hEScells, as described later in this disclosure. In coculture with pPScells, feeder cells are typically inactivated by irradiation ortreatment with an anti-mitotic agent such as mitomycin c, to preventthem from outgrowing the cells they are supporting. For use in producingconditioned medium, inactivation of the cells may be optional, anddepends in part on mechanical aspects of medium production.

[0050] pPS cell populations are said to be “essentially free” of feedercells if the cells have been grown through at least one round aftersplitting in which fresh feeder cells are not added to support thegrowth of the pPS. It is recognized that if a previous culturecontaining feeder cells is used as a source of pPS for the culture towhich fresh feeders are not added, there will be some feeder cells thatsurvive the passage. For example, hES cells are often cultured in a 9.6cm² well on a surface of ˜375,000 primary irradiated embryonicfibroblasts near confluence. By the end of the culture, perhaps 150,000feeder cells are still viable, and will be split and passaged along withhES that have proliferated to a number of ˜1 to 1.5 million. After a 1:6split, the hES cells generally resume proliferation, but the fibroblastswill not grow and only a small proportion will be viable by the end of˜6 days of culture. This culture is essentially free of feeder cells,with compositions containing less than about 5% feeder cells.Compositions containing less than 1%, 0.2%, 0.05%, or 0.01% feeder cells(expressed as % of total cells in the culture) are increasingly morepreferred.

[0051] Whenever a culture or cell population is referred to in thisdisclosure as “feeder-free”, what is meant is that the composition isessentially free of feeder cells according to the preceding definition,subject only to further constraints if explicitly required.

[0052] A “growth environment” is an environment in which cells ofinterest will proliferate or differentiate in vitro. Features of theenvironment include the medium in which the cells are cultured, thetemperature, the partial pressure of O₂ and CO₂, and a supportingstructure (such as a substrate on a solid surface) if present.

[0053] A “nutrient medium” is a medium for culturing cells containingnutrients that promote proliferation. The nutrient medium may containany of the following in an appropriate combination: isotonic saline,buffer, amino acids, antibiotics, serum or serum replacement, andexogenously added factors. A “conditioned medium” is prepared byculturing a first population of cells in a medium, and then harvestingthe medium. The conditioned medium (along with anything secreted intothe medium by the cells) may then be used to support the growth of asecond population of cells.

[0054] “Embryoid bodies” is a term of art synonymous with “aggregatebodies”. The terms refer to aggregates of differentiated andundifferentiated cells that appear when pPS cells overgrow in plated orsuspension cultures.

[0055] “Restricted developmental lineage cells” are cells derived fromembryonic tissue, typically by differentiation or partialdifferentiation of pPS cells. These cells are capable of proliferatingand differentiating into several different cell types, but the range oftheir repertory is restricted. Examples are hematopoietic cells, whichare pluripotent for blood cell types, and hepatocyte progenitors, whichare pluripotent for sinusoidal endothelial cells, hepatocytes, andpotentially other liver cells. Another example is neural restrictedcells, which can generate glial cell precursors that progress tooligodendrocytes and astrocytes, and neuronal precursors that progressto neurons.

[0056] A cell is said to be “genetically altered”, “transfected”, or“genetically transformed” when a polynucleotide has been transferredinto the cell by any suitable means of artificial manipulation, or wherethe cell is a progeny of the originally altered cell that has inheritedthe polynucleotide. The polynucleotide will often comprise atranscribable sequence encoding a protein of interest, which enables thecell to express the protein at an elevated level. Also included aregenetic alterations by any means that result in functionally altering orabolishing the action of an endogenous gene. The genetic alteration issaid to be “inheritable” if progeny of the altered cell has the samealteration.

[0057] A “cell line” is a population of cells that can be propagated inculture through at least 10 passages. The population can bephenotypically homogeneous, or the population can be a mixture ofmeasurably different phenotypes. Characteristics of the cell line arethose characteristics of the population as a whole that are essentiallyunaltered after 10 passages.

[0058] A cell is described as “telomerized” if it has been geneticallyaltered with a nucleic acid encoding a telomerase reverse transcriptase(TERT) of any species in such a manner that the TERT is transcribed andtranslated in the cell. The term also applies to progeny of theoriginally altered cell that have inherited the ability to express theTERT encoding region at an elevated level. The TERT encoding sequence istypically taken or adapted from a mammalian TERT gene, exemplified byhuman and mouse TERT, as indicated below.

[0059] A cell line is described as “permanent” or “immortalized” if ithas at least one of the following properties: 1) it has been geneticallyaltered for elevated expression of telomerase reverse transcriptase(TERT), detectable, for example, as increased telomerase activity inTRAP assay; 2) for cell lines otherwise capable of no more than 15population doublings, it has been genetically altered to extend itsreplicative capacity under suitable culture conditions to at least 20population doublings; or 3) for cell lines otherwise capable of morethan 15 population doublings, it has been genetically altered tosignificantly extend the replicative capacity of the cell line undertypical culture conditions. It is understood that cells meeting thisdefinition include not only the original genetically altered cells, butalso all progeny of such cells that meet the listed criteria.

[0060] The term “antibody” as used in this disclosure refers to bothpolyclonal and monoclonal antibody. The ambit of the term deliberatelyencompasses not only intact immunoglobulin molecules, but also fragmentsand derivatives of immunoglobulin molecules (such as single chain Fvconstructs), and fragments and derivatives of immunoglobulin equivalentssuch as T-cell receptors, as may be prepared by techniques known in theart, and retaining the desired antigen binding specificity.

[0061] General Techniques

[0062] For further elaboration of general techniques useful in thepractice of this invention, the practitioner can refer to standardtextbooks and reviews in cell biology, tissue culture, and embryology.Included are Teratocarcinomas and embryonic stem cells: A practicalapproach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide toTechniques in Mouse Development (P. M. Wasserman et al., eds., AcademicPress 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles,Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic StemCells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., al.,1993). Differentiation of stem cells is reviewedin Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod.Fertil. Dev. 10:31, 1998.

[0063] Methods in molecular genetics and genetic engineering aredescribed generally in the current editions of Molecular Cloning: ALaboratory Manual, (Sambrook et al.); Oligonucleotide Synthesis (M. J.Gait, ed.,); Animal Cell Culture (R. I. Freshney, ed.); Gene TransferVectors for Mammalian Cells (Miller & Calós, eds.); Current Protocols inMolecular Biology and Short Protocols in Molecular Biology, 3rd Edition(F. M. Ausubel et al., eds.); and Recombinant DNA Methodology (R. Wued., Academic Press). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, and ClonTech.

[0064] For general techniques involved in preparation of mRNA and cDNAlibraries and their analysis, those skilled in the art have access toRNA Methodologies: A Laboratory Guide for Isolation and Characterization(R. E. Farrell, Academic Press, 1998); cDNA Library Protocols (Cowell &Austin, eds., Humana Press); Functional Genomics (Hunt & Livesey, eds.,2000); and the Annual Review of Genomics and Human Genetics (E Lander,ed., published yearly by Annual-Reviews). General techniques used inraising, purifying and modifying antibodies, and the design andexecution of immunoassays including immunocytochemistry, the reader isreferred to Handbook of Experimental Immunology (Weir & Blackwell,eds.); Current Protocols in Immunology (Coligan et al., eds.); andMethods of Immunological Analysis (Masseyeff et al., eds., Weinheim: VCHVerlags GmbH).

[0065] General techniques in cell culture and media collection areoutlined in Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin.Biotechnol. 8:148, 1997); Serum-free Media (K. Kitano, Biotechnology17:73, 1991); Large Scale Mammalian Cell Culture (Curr. Opin.Biotechnol. 2:375, 1991); and Suspension Culture of Mammalian Cells(Birch et al., Bioprocess Technol. 19:251,1990).

[0066] Sources of pluripotent stem cells

[0067] Suitable source cells for culturing and differentiation accordingto this invention include established lines of pluripotent cells derivedfrom tissue formed after gestation. Exemplary primary tissue sources areembryonic tissue (such as a blastocyst), or fetal tissue taken any timeduring gestation, typically but not necessarily before 10 weeksgestation. Non-limiting exemplars are established lines of primateembryonic stem (ES) and embryonic germ (EG) cells. Also contemplated isuse of the techniques of this disclosure during the initialestablishment or stabilization of such cells, in which case the sourcecells would be primary pluripotent cells taken directly from the tissueslisted.

[0068] Media and Feeder Cells

[0069] Media for isolating and propagating pPS cells can have any ofseveral different formulas, as long as the cells obtained have thedesired characteristics, and can be propagated further. Suitable sourcesare as follows: Dulbecco's modified Eagles medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagles medium (KO DMEM), Gibco #10829-018; 200 mM L-glutamine, Gibco # 15039-027; non-essential aminoacid solution, Gibco 11140-050; ,β-mercaptoethanol, Sigma # M7522; humanrecombinant basic fibroblast growth factor (bFGF), Gibco # 13256-029.Exemplary serum-containing ES medium is made with 80% DMEM (typically KODMEM), 20% defined fetal bovine serum (FBS) not heat inactivated, 0.1 mMnon-essential amino acids, 1 mM L-glutamine, and 0.1 mMβ-mercaptoethanol. The medium is filtered and stored at 4° C. Serum-freeES medium is made with 80% KO DMEM, 20% serum replacement, 0.1 mMnon-essential amino acids, 1 mM L-glutamine, and 0.1 mMβ-mercaptoethanol. Not all serum replacements work; an effective serumreplacement is Gibco # 10828-028 (proprietary formula; productobtainable from the manufacturer). The medium is filtered and stored at4° C. Just before use, human bFGF is added to a final concentration of 4ng/mL.

[0070] pPS cells are typically cultured on a layer of feeder cells thatsupport the pPS cells in various ways, such as the production of solublefactors that promote pPS cell survival or proliferation, or inhibitdifferentiation. Feeder cells are typically fibroblast type cells, oftenderived from embryonic or fetal tissue. A frequently used source ismouse embryo. Useful feeder cell lines have been obtained by obtainingembryonic fibroblasts, transfecting them to express telomerase, and thenpassaging them or freezing them for future use. The cell lines areplated to near confluence, irradiated to prevent proliferation, and usedto support pPS cell cultures.

[0071] In one illustration, pPS cells are first derived and supported onprimary embryonic fibroblasts. Mouse embryonic fibroblasts (mEF) can beobtained from outbred CF1 mice (SASCO) or other suitable strains. Theabdomen of a mouse at 13 days of pregnancy is swabbed with 70% ethanol,and the decidua is removed into phosphate buffered saline (PBS). Embryosare harvested; placenta, membranes, and soft tissues are removed; andthe carcasses are washed twice in PBS. They are then transferred tofresh 10 cm bacterial dishes containing 2 mL trypsin/EDTA, and finelyminced. After incubating 5 min at 37° C., the trypsin is inactivatedwith 5 mL DMEM containing 10% FBS, and the mixture is transferred to a15 mL conical tube. Debris is allowed to settle for 2 min, thesupernatant is made up to a final volume of 10 mL, and plated onto a 10cm tissue culture plate or T75 flask. The flask is incubated undisturbedfor 24 h, after which the medium is replaced. When flasks are confluent(˜2-3 d), they are split 1:2 into new flasks.

[0072] Feeder cells are propagated in mEF medium, containing 90% DMEM(Gibco # 11965-092), 10% FBS (Hyclone # 30071-03), and 2 mM glutamine.mEFs are propagated in T150 flasks (Corning # 430825), splitting thecells 1:2 every other day with trypsin, keeping the cells subconfluent.To prepare the feeder cell layer, cells are irradiated at a dose toinhibit proliferation but permit synthesis of important factors thatsupport hES cells (˜4000 rads gamma irradiation). Six-well cultureplates (such as Falcon # 304) are coated by incubation at 37° C. with 1mL 0.5% gelatin per well overnight, and plated with ˜375,000 irradiatedmEFs per well. Feeder cell layers are used 5 h to 4 days after plating.The medium is replaced with fresh hES medium just before seeding pPScells.

[0073] Preparation of human embryonic stem (hES) cells

[0074] Human embryonic stem (hES) cells can be prepared as described byThomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr.Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. USA 92:7844,1995).

[0075] Briefly, human blastocysts are obtained from human in vivopreimplantation embryos. Alternatively, in vitro fertilized (IVF)embryos can be used, or one cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Human embryosare cultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner etal., Fertil. Steril. 69:84, 1998). Blastocysts that develop are selectedfor ES cell isolation. The zona pellucida is removed from blastocysts bybrief exposure to pronase (Sigma). The inner cell masses are isolated byimmunosurgery, in which blastocysts are exposed to a 1:50 dilution ofrabbit anti-human spleen cell antiserum for 30 minutes, then washed for5 minutes three times in DMEM, and exposed to a 1:5 dilution of Guineapig complement (Gibco) for 3 min (see Solter et al., Proc. Natl. Acad.Sci. USA 72:5099, 1975). After two further washes in DMEM, lysedtrophectoderm cells are removed from the intact inner cell mass (ICM) bygentle pipetting, and the ICM plated on mEF feeder layers.

[0076] After 9 to 15 days, inner cell mass-derived outgrowths aredissociated into clumps either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Dissociated cells are replated on mEFfeeder layers in fresh ES medium, and observed for colony formation.Colonies demonstrating undifferentiated morphology are individuallyselected by micropipette, mechanically dissociated into clumps, andreplated. ES-like morphology is characterized as compact colonies withapparently high nucleus to cytoplasm ratio and prominent nucleoli.Resulting ES cells are then routinely split every 1-2 weeks by brieftrypsinization, exposure to Dulbecco's PBS (without calcium or magnesiumand with 2 mM EDTA), exposure to type IV collagenase (˜200 U/mL; Gibco)or by selection of individual colonies by micropipette. Clump sizes ofabout 50 to 100 cells are optimal.

[0077] Preparation of human embryonic germ (hEG) cells

[0078] Human Embryonic Germ (hEG) cells can be prepared from primordialgerm cells present in human fetal material taken about 8-11 weeks afterthe last menstrual period. Suitable preparation methods are described inShamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S.Pat. No. 6,090,622.

[0079] Briefly, genital ridges are rinsed with isotonic buffer, thenplaced into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) andcut into <1 mm³ chunks. The tissue is then pipetted through a 100 μL tipto further disaggregate the cells. It is incubated at 37° C. for ˜5 min,then ˜3.5 mL EG growth medium is added. EG growth medium is DMEM, 4500mg/L D-glucose, 2200 mg/L mM sodium bicarbonate; 15% ES qualified fetalcalf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL);1000-2000 U/mL human recombinant leukemia inhibitory factor (LIF,Genzyme); 1-2 ng/ml human recombinant basic fibroblast growth factor(bFGF, Genzyme); and 10 μM forskolin (in 10% DMSO). In an alternativeapproach, EG cells are isolated using hyaluronidase/collagenase/DNAse.Gonadal anlagen or genital ridges with mesenteries are dissected fromfetal material, the genital ridges are rinsed in PBS, then placed in 0.1ml HCD digestion solution (0.01% hyaluronidase type V, 0.002% DNAse I,0.1% collagenase type IV, all from Sigma prepared in EG growth medium).Tissue is minced and incubated 1 h or overnight at 37° C., resuspendedin 1-3 mL of EG growth medium, and plated onto a feeder layer.

[0080] Ninety-six well tissue culture plates are prepared with asub-confluent layer of feeder cells cultured for 3 days in modified EGgrowth medium free of LIF, bFGF or forskolin, inactivated with 5000 radγ-irradiation. Suitable feeders are STO cells (ATCC Accession No. CRL1503). ˜0.2 mL of primary germ cell (PGC) suspension is added to each ofthe wells. The first passage is conducted after 7-10 days in EG growthmedium, transferring each well to one well of a 24-well culture dishpreviously prepared with irradiated STO mouse fibroblasts. The cells arecultured with daily replacement of medium until cell morphologyconsistent with EG cells are observed, typically after 7-30 days or 1-4passages.

[0081] Propagation of pPS cells

[0082] pPS cells can be propagated continuously in culture, using acombination of culture conditions that support proliferation withoutpromoting differentiation. It has been determined that hES cells can begrown without differentiation, even in the absence of feeder cells. Forfeeder-free culture, it is beneficial to provide a compatible culturesurface (the substrate), and a nutrient medium that supplies some of theinfluences provided by the feeder cells.

[0083] Particularly suitable as a substrate for feeder-free pPS cultureare extracellular matrix components (derived from basement membrane, orforming part of adhesion molecule receptor-ligand couplings). Acommercial preparation is available from Becton Dickenson under the nameMatrigel®, and can be obtained in regular or Growth Factor Reducedformulation. Both formulations are effective. Matrigel® is a solublepreparation from Engelbreth-Holm-Swarm tumor cells that gels at roomtemperature to form a reconstituted basement membrane.

[0084] Other extracellular matrix components and component mixtures aresuitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparan sulfate, and the like, alone or in variouscombinations. Laminins are major components of all basal laminae invertebrates, which interact with integrin heterodimers such as α6β1 andα6β4 (specific for laminins) and other heterodimers (that cross-reactwith other matrices). Using culture conditions illustrated in theexamples, collagen IV supports hES cell growth, while collagen I doesnot. Substrates that can be tested using the experimental proceduresdescribed herein include not only other extracellular matrix components,but also polyamines (such as poly-ornithine, poly-lysine), and othercommercially available coatings.

[0085] The pluripotent cells are plated onto the substrate in a suitabledistribution and in the presence of a medium that promotes cellsurvival, propagation, and retention of the desirable characteristics.These characteristics benefit from careful attention to the seedingdistribution. One feature of the distribution is the plating density. Ithas been found that plating densities of at least ˜15,000 cells cm⁻²promote survival and limit differentiation. Typically, a plating densityof between about 90,000 cm⁻² and about 170,000 cm⁻² is used.

[0086] Another feature is the dispersion of cells. The propagation ofmouse stem cells involves dispersing the cells into a single-cellsuspension (Robinson, Meth. Mol. Biol. 75:173, 1997 at page 177). Incontrast, passaging primate PS cells has previously thought to requirekeeping the cells together in small clusters. Enzymatic digestion ishalted before cells become completely dispersed (say, ˜5 min withcollagenase IV). The plate is then scraped gently with a pipette, andthe cells are triturated with the pipette until they are suspended asclumps of adherent cells, about 10-2000 cells in size. The clumps arethen plated directly onto the substrate without further dispersal.

[0087] It has been discovered that primate PS cells can be passagedbetween feeder-free cultures as a finer cell suspension, providing thatan appropriate enzyme and medium are chosen, and the plating density issufficiently high. By way of illustration, confluent human embryonicstem cells cultured in the absence of feeders are removed from theplates by incubating with a solution of 0.05% (wt/vol) trypsin (Gibco)and 0.053 mM EDTA for 5-15 min at 37° C. With the use of pipette, theremaining cells in the plate are removed and the cells are trituratedwith the pipette until the cells are dispersed into a suspensioncomprising single cells and some small clusters. The cells are thenplated at densities of 50,000-200,000 cells/cm² to promote survival andlimit differentiation. The phenotype of ES cells passaged by thistechnique is similar to what is observed when cells are harvested asclusters by collagen digestion. As another option, the cells can beharvested without enzymes before the plate reaches confluence. The cellsare incubated ˜5 min in a solution of 0.5 mM EDTA alone in PBS, washedfrom the culture vessel, and then plated into a new culture withoutfurther dispersal.

[0088] pPS cells plated in the absence of fresh feeder cells benefitfrom being cultured in a nutrient medium. The medium will generallycontain the usual components to enhance cell survival, includingisotonic buffer, essential minerals, and either serum or a serumreplacement of some kind. Particularly beneficial is a medium that hasbeen conditioned to supply some of the elements otherwise provided byfeeder cells.

[0089] Feeder cells typically contain fibroblast type cells. Primaryembryonic or fetal feeder cell cultures are a mixed population of cells,containing cells that have morphology of fibroblasts and of early muscleand neuronal cells. Different cells in the population may play differentroles in supporting pPS culture, and the distribution and character ofthe culture may change.

[0090] As an alternative to primary mouse fibroblast cultures,conditioned medium can be prepared from other cell types, such asestablished cell lines. More permanent feeder cell lines can bedeveloped for producing medium according to this invention usingembryonic fibroblasts from a non-human species such as a mouse,genetically altered with an immortalizing gene, such as a gene thatexpresses telomerase.

[0091] It has also been discovered that cells with particularcharacteristics differentiated from human embryo derived cells can beused to support culture of undifferentiated pPS cells. Certainfibroblast-like cells or mesenchymal cells derived from human embryocells have this property, and can be identified according to the assaydescribed earlier. An exemplary method for obtaining suitable cellsinvolves differentiating a culture of pPS cells (such as hES cells).Differentiated cells with a particular phenotype are selected fromamongst the mixed differentiated cell population, and medium conditionedby culturing with the selected cells is tested for its ability tosupport growth of pPS cells in a culture environment essentially free offeeder cells. As illustrated in the examples below, medium that has beenconditioned for 1-2 days is typically used to support pPS cell culturefor 1-2 days, and then exchanged. Conditioned medium is used to supportpPS cells undiluted, or titrated to an effective level of dilution. Theconditioned medium can be supplemented before use with additional growthfactors that benefit pPS cell culture. It is often beneficial to addgrowth factors such as bFGF or FGF-4 to the medium both beforeconditioning, and then again before using the medium to support thegrowth of pPS cells.

[0092] It should be recognized that each of the conditions describedhere can be optimized independently, and certain combinations ofconditions will prove effective upon further testing. Such optimizationis a matter of routine experimentation, and does not depart from thespirit of the invention provided in this disclosure.

[0093] Characteristics of pPS cells

[0094] Human ES cells have the characteristic morphological features ofundifferentiated stem cells. In the two dimensions of a standardmicroscopic image, hES cells have high nuclear/cytoplasmic ratios in theplane of the image, prominent nucleoli, and compact colony formationwith poorly discernable cell junctions. Cell lines can be karyotypedusing a standard G-banding technique (available at many clinicaldiagnostics labs that provides routine karyotyping services, such as theCytogenetics Lab at Oakland Calif.) and compared to published humankaryotypes. It is desirable to obtain cells that have a “normalkaryotype”, which means that the cells are euploid, wherein all humanchromosomes are present and are not noticeably altered.

[0095] hES and hEG cells can also be characterized by expressed cellmarkers. In general, the tissue-specific markers discussed in thisdisclosure can be detected using a suitable immunological technique—suchas flow cytometry for membrane-bound markers, immunocytochemistry forintracellular markers, and enzyme-linked immunoassay, for markerssecreted into the medium. The expression of protein markers can also bedetected at the mRNA level by reverse transcriptase-PCR usingmarker-specific primers. See U.S. Pat. No. 5,843,780 for furtherdetails.

[0096] Stage-specific embryonic antigens (SSEA) are characteristic ofcertain embryonic cell types. Antibodies for SSEA-1, SSEA-3 and SSEA-4are available from the Developmental Studies Hybridoma Bank of theNational Institute of Child Health and Human Development (Bethesda Md.).Other useful markers are detectable using antibodies designated Tra-1-60and Tra-1-81 (Andrews et al., Cell Lines from Human Germ Cell Tumors, inE. J. Robertson, 1987, supra). Mouse ES cells can be used as a positivecontrol for SSEA-1, and as a negative control for SSEA-4, Tra-1-60, andTra-1-81. SSEA-4 is consistently present on human embryonal carcinoma(hEC) cells. Differentiation of pPS cells in vitro results in the lossof SSEA-4, Tra-1-60, and Tra-1-81 expression and increased expression ofSSEA-1. SSEA-1 is also found on hEG cells. pPS cells can also becharacterized by the presence of alkaline phosphatase activity, whichcan be detected by fixing the cells with 4% paraformaldehyde, and thendeveloping with Vector Red as a substrate, as described by themanufacturer (Vector Laboratories, Burlingame Calif.). Expression ofhTERT and OCT-4 (detectable by RT-PCR) and telomerase activity(detectable by TRAP assay) are also characteristic of many types ofundifferentiated pPS cells.

[0097] Where it is desirable to increase the replicative capacity of pPScells, or cells differentiated from them, they can be immortalized ortelomerized (either before or after differentiation) using the methodsdescribed below.

[0098] Differentiation of propagated pPS cells

[0099] This invention provides a new system for differentiating pPScells into committed precursor cells or fully differentiated cellswithout forming embryoid bodies as an intermediate step.

[0100] Culturing embryoid bodies according to traditional methods arereported in O'Shea, Anat. Rec. (New Anat. 257:323, 1999). pPS cells arecultured in a manner that permits aggregates to form, for which manyoptions are available: for example, by overgrowth of a donor pPS cellculture, or by culturing pPS cells in suspension in culture vesselshaving a substrate with low adhesion properties which allows EBformation. pPS cells are harvested by brief collagenase digestion,dissociated into clusters, and plated in non-adherent cell cultureplates. The aggregates are fed every few days, and then harvested aftera suitable period, typically 4-8 days.

[0101] The cells can then be cultured in a medium and/or on a substratethat promotes enrichment of cells of a particular lineage. The substratecan comprise matrix components such as Matrigel® (Becton Dickenson),laminin, collagen, gelatin, or matrix produced by first culturing amatrix-producing cell line (such as a fibroblast or endothelial cellline), and then lysing and washing in such a way that the matrix remainsattached to the surface of the vessel. Embryoid bodies comprise aheterogeneous cell population, potentially having an endoderm exterior,and a mesoderm and ectoderm interior.

[0102] The Direct Differentiation method

[0103] It has now been discovered that pPS cells can be differentiatedinto committed precursor cells or terminally differentiated cellswithout forming embryoid bodies or aggregates as an intermediate step.

[0104] Briefly, a suspension of undifferentiated pPS cells is prepared,and then plated onto a solid surface that promotes differentiation. Ingeneral, cultures of pPS cells are typically harvested when they haveproliferated to an adequate density, but not to the point ofover-confluence, because the cells will differentiate in an uncontrolledfashion if allowed to overgrow. A suitable suspension can be prepared byincubating the culture dish with Collagenase IV for about 5-20 min, andthen scraping the cells from the dish. The cells can be dissociated, forexample, by triturating in a pipette. For many types of differentiation,it is recommended that the cells not be completely dissociated, so thatthe majority of pPS is in clumps of about 10 to 200 cells.

[0105] The suspension is then plated onto a substrate that promotesregulated differentiation into committed precursor cells. Suitablesubstrates include glass or plastic surfaces that are adherent. Forexample, glass coverslips can be coated with a poly-cationic substance,such as a polyamine like poly-lysine, poly-ornithine, or otherhomogeneous or mixed polypeptides or other polymers with a predominantpositive charge. The cells are then cultured in a suitable nutrientmedium that is adapted to promote differentiation towards the desiredcell lineage.

[0106] In some instances, differentiation is promoted by withdrawingserum or serum replacement from the culture medium. This can be achievedby substituting a medium devoid of serum and serum replacement, forexample, at the time of replating, by withdrawing one or more componentsof the medium that promotes growth of undifferentiated cells or inhibitsdifferentiation. Examples include certain growth factors, mitogens,leukemia inhibitory factor (LIF), fibroblast growth factors such asbFGF, and other components in conditioned medium. The new medium is saidto be “essentially free” of these components when it contains <5% andpreferably <1% of the usual concentration of the component used inculturing the cells in an undifferentiated form.

[0107] In some instances, differentiation is promoted by adding a mediumcomponent that promotes differentiation towards the desired celllineage, or inhibits the growth of cells with undesired characteristics.For example, to generate cells committed to neural or glial lineages,the medium can include any of the following factors or mediumconstituents in an effective combination: Brain derived neurotrophicfactor (BDNF), neutrotrophin-3 (NT-3), NT-4, epidermal growth factor(EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF),retinoic acid (RA), sonic hedgehog, FGF-8, ascorbic acid, forskolin,fetal bovine serum (FBS), and bone morphogenic proteins (BMPs). Otherexemplary factors are listed in Example 5.

[0108] Under appropriate conditions, the direct differentiation methodprovides a cell population that is less heterogeneous than what istypically found in embryoid body derived cells. Unless explicitlyindicated otherwise, the method can include a small degree ofovergrowth, aggregate formation, or formation of occasional embryoidbody-like structure—however, this is a collateral occurrence, and notrequired for differentiation of the cells into the committed precursoror terminally differentiated cell population desired. Typically, lessthan ˜10% of the differentiated cell population will be progeny of cellsthat grew out of embryoid bodies, with levels of less than ˜3% or ˜1%being achievable in certain circumstances.

[0109] General principals for obtaining tissue cells from pluripotentstem cells are reviewed in Pedersen (Reprod. Fertil. Dev. 6:543, 1994),and U.S. Pat. No.6,090,622. For neural progenitors, neural restrictivecells and glial cell precursors, see Bain et al., Biochem. Biophys. Res.Commun. 200:1252, 1994;Trojanowski et al., Exp. Neurol. 144:92, 1997;Wojcik et al., Proc. Natl. Acad. Sci. USA 90:1305-130; Mujtaba et al.,Dev. Biol. 214:113, 1999; and U.S. Pat. Nos. 5,851,832, 5,928,947,5,766,948, and 5,849,553. For cardiac muscle and cardiomyocytes see Chenet al., Dev. Dynamics 197:217, 1993 and Wobus et al., Differentiation48:173, 1991. For hematopoietic progenitors, see Burkert et al., NewBiol. 3:698, 1991 and Biesecker et al., Exp. Hematol. 21:774, 1993. U.S.Pat. No. 5,773,255 relates to glucose-responsive insulin secretingpancreatic beta cell lines. U.S. Pat. No. 5,789,246 relates tohepatocyte precursor cells. Other progenitors of interest include butare not limited to chondrocytes, osteoblasts, retinal pigment epithelialcells, fibroblasts, skin cells such as keratinocytes, dendritic cells,hair follicle cells, renal duct epithelial cells, smooth and skeletalmuscle cells, testicular progenitors, and vascular endothelial cells.

[0110] Scientists at Geron Corporation have discovered that culturingpPS cells or embryoid body cells in the presence of ligands that bindgrowth factor receptors promotes enrichment for neural precursor cells.The growth environment may contain a neural cell supportiveextracellular matrix, such as fibronectin. Suitable growth factorsinclude but are not limited to EGF, bFGF, PDGF, IGF-1, and antibodies toreceptors for these ligands. Cofactors such as retinoic acid may also beincluded. The cultured cells may then be optionally separated based onwhether they express a marker such as A2B5. Under the appropriatecircumstances, populations of cells enriched for expression of the A2B5marker may have the capacity to generate both neuronal cells (includingmature neurons), and glial cells (including astrocytes andoligodendrocytes.

[0111] Optionally, the cell populations are further differentiated, forexample, by culturing in a medium containing an activator of cAMP.Factors useful in the direct differentiation method for producingneurons are explored in Example 5, below. Markers for identifying celltypes include β-tubulin III or microtubule-associated protein 2 (MAP-2),characteristic of neurons; glial fibrillary acidic protein (GFAP),present in astrocytes; galactocerebroside (GaIC) or myelin basic protein(MBP); characteristic of oligodendrocytes; OCT-4, characteristic ofundifferentiated hES cells; Nestin or Musashi, characteristic of neuralprecursors and other cells; and both A2B5 and NCAM, which appear onpopulations of neural precursors differentiated from pPS cells.

[0112] Scientists at Geron Corporation have also discovered thatculturing pPS cells or embryoid body cells in the presence of ahepatocyte differentiation agent promotes enrichment for hepatocyte-likecells. The growth environment may contain a hepatocyte supportiveextracellular matrix, such as collagen or Matrigel®. Suitabledifferentiation agents include various isomers of butyrate and theiranalogs, exemplified by n-butyrate. The cultured cells are optionallycultured simultaneously or sequentially with a hepatocyte maturationfactor, such as an organic solvent like dimethyl sulfoxide (DMSO); amaturation cofactor such as retinoic acid; or a cytokine or hormone suchas a glucocorticoid, epidermal growth factor (EGF), insulin,transforming growth factors (TGF-α and TGF-β), fibroblast growth factors(FGF), heparin, hepatocyte growth factors (HGF), interleukins (IL-1 andIL-6), insulin-like growth factors (IGF-I and IGF-II), andheparin-binding growth factors (HBGF-1).

[0113] Characteristics of differentiated cells

[0114] Cells can be characterized according to a number of phenotypiccriteria. The criteria include but are not limited to characterizationof morphological features, detection or quantitation of expressed cellmarkers and enzymatic activity, and determination of the functionalproperties of the cells in vivo.

[0115] Markers of interest for neural cells include β-tubulin IIII orneurofilament, characteristic of neurons; glial fibrillary acidicprotein (GFAP), present in astrocytes; galactocerebroside (GaIC) ormyelin basic protein (MBP); characteristic of oligodendrocytes; Oct-4,characteristic of undifferentiated hES cells; nestin, characteristic ofneural precursors and other cells. A2B5 and NCAM are characteristic ofglial progenitors and neural progenitors, respectively. Cells can alsobe tested for secretion of characteristic biologically activesubstances. For example, GABA-secreting neurons can be identified byproduction of glutamic acid decarboxylase or GABA. Dopaminergic neuronscan be identified by production of dopa decarboxylase, dopamine, ortyrosine hydroxylase.

[0116] Markers of interest for liver cells include α-fetoprotein (liverprogenitors); albumin, α₁-antitrypsin, glucose-6-phosphatase, cytochromep450 activity, transferrin, asialoglycoprotein receptor, and glycogenstorage (hepatocytes); CK7, CK19, and γ-glutamyl transferase (bileepithelium). It has been reported that hepatocyte differentiationrequires the transcription factor HNF-4α(Li et al., Genes Dev. 14:464,2000). Markers independent of HNF-4α expression include α₁-antitrypsin,α-fetoprotein, apoE, glucokinase, insulin growth factors 1 and 2, IGF-1receptor, insulin receptor, and leptin. Markers dependent on HNF-4αexpression include albumin, apoAI, apoAII, apoB, apoCIII, apoCII,aldolase B, phenylalanine hydroxylase, L-type fatty acid bindingprotein, transferrin, retinol binding protein, and erythropoietin (EPO).Hepatocyte lineage cells differentiated from pPS cells will typicallydisplay at least three of the following markers: α₁-antitrypsin (AAT)synthesis, albumin synthesis, asialoglycoprotein receptor (ASGR)expression, absence of a-fetoprotein, evidence of glycogen storage,evidence of cytochrome p450 activity, and evidence ofglucose-6-phosphatase activity.

[0117] Markers of interest for other cell types include the following.For cardiomyocytes: GATA-4, Nkx2.5, cardiac troponin I, ANF, α-cardiacmyosin heavy chain (α-MHC), actin, or ventricular myosin light chain 2(MLC-2v). See Wobus et al., J. Mol. Cell Cardiol. 29:1525, 1997. Forskeletal muscle: myoD, myogenin, and myf-5. For endothelial cells: PECAM(platelet endothelial cell adhesion molecule), Flk-1, tie-1, tie-2,vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. For smoothmuscle cells: specific myosin heavy chain. For pancreatic cells, pdx andinsulin secretion. For hematopoietic cells and their progenitors:GATA-1, CD34, β-major globulin, and β-major globulin like gene βH1.

[0118] Certain tissue-specific markers listed in this disclosure orknown in the art can be detected by immunological techniques—such asflow immunocytochemistry for cell-surface markers, immunohistochemistry(for example, of fixed cells or tissue sections) for intracellular orcell-surface markers, Western blot analysis of cellular extracts, andenzyme-linked immunoassay, for cellular extracts or products secretedinto the medium. The expression of tissue-specific gene products canalso be detected at the mRNA level by Northern blot analysis, dot-blothybridization analysis, or by reverse transcriptase initiated polymerasechain reaction (RT-PCR) using sequence-specific primers in standardamplification methods. Sequence data for the particular markers listedin this disclosure can be obtained from public databases such as GenBank(URL www.ncbi.nlm.nih.gov:80/entrez). Expression of tissue-specificmarkers as detected at the protein or mRNA level is considered positiveif the level is at least 2-fold, and preferably more than 10- or 50-foldabove that of a control cell, such as an undifferentiated pPS cell, afibroblast, or other unrelated cell type.

[0119] The system provided by this invention allows production of arelatively uniform cell population, without the complexity of cellsoften obtained by forming embryoid bodies. Cell populations derived bydirect differentiation may be 50%, 75%, 90%, or 98% homogeneous in termsof morphological characteristics of the desired cell type, or expressionof any of the markers indicated above. They may also be relativelydevoid of undesired cell types, such as endothelial cells, mesenchymalcells, fibroblasts, smooth muscle cells, cells expressing α-myosin heavychain, or other particular cell types of the endoderm, mesoderm, orectoderm.

[0120] Modifying differentiated cells

[0121] Differentiated cells of this invention can be genetically alteredin a manner that permits the genetic alteration to be either transient,or stable and inheritable as the cells divide. Undifferentiated cellscan be genetically altered and then differentiated into the desiredphenotype, or the cells can be differentiated first before geneticalteration. Where the pPS cells are genetically altered beforedifferentiation, the genetic alteration can be performed on a permanentfeeder cell line that has resistance genes for drugs used to select fortransformed cells, or on pPS cells grown in feeder-free culture.

[0122] Suitable methods for transferring vector plasmids into hES cellsinclude lipid/DNA complexes, such as those described in U.S. Pat. Nos.5,578,475; 5,627,175; 5,705,308; 5,744,335; 5,976,567; 6,020,202; and6,051,429. Suitable reagents include lipofectamine, a 3:1 (w/w) liposomeformulation of the poly-cationic lipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA) (Chemical Abstracts Registry name:N-[2-(2,5-bis[(3-aminopropyl)amino]-1-oxpentyl}amino)ethyl]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanaminiumtrifluoroacetate), and the neutral lipid dioleoylphosphatidylethanolamine (DOPE) in membrane filtered water. Exemplary isthe formulation Lipofectamine 2000™ (available from Gibco/LifeTechnologies # 11668019). Other reagents include: FuGENE™ 6 TransfectionReagent (a blend of lipids in non-liposomal form and other compounds in80% ethanol, obtainable from Roche Diagnostics Corp. # 1814443); andLipoTAXI™ transfection reagent (a lipid formulation from InvitrogenCorp., #204110). Suitable viral vector systems for producing hES cellswith stable genetic alterations are based on adenovirus and retrovirus,and may be prepared using commercially available virus components.

[0123] For therapeutic use, it is usually desirable that differentiatedcell populations be substantially free of undifferentiated pPS cells.One way of depleting undifferentiated stem cells from the population isto transfect them with a vector in which an effector gene under controlof a promoter that causes preferential expression in undifferentiatedcells. Suitable promoters include the TERT promoter and the OCT-4promoter. The effector gene may be directly lytic to the cell (encoding,for example, a toxin or a mediator of apoptosis). Alternatively, theeffector gene may render the cell susceptible to toxic effects of anexternal agent, such as an antibody or a prodrug. Exemplary is a herpessimplex thymidine kinase (tk) gene, which causes cells in which it isexpressed to be susceptible to ganciclovir. Suitable TERT promoter tkconstructs are provided in WO 98/14593 (Morin et al.).

[0124] Increasing replicative capacity of differentiated cells

[0125] It is desirable that certain differentiated cells have theability to replicate in certain drug screening and therapeuticapplications. Cells can optionally be telomerized to increase theirreplication potential, either before or after they progress torestricted developmental lineage cells or terminally differentiatedcells. pPS cells that are telomerized may be taken down thedifferentiation pathway described earlier; or differentiated cells canbe telomerized directly.

[0126] Before and after telomerization, telomerase activity andexpression of hTERT gene product can be determined using commerciallyavailable reagents and established methods. For example, pPS cells areevaluated for telomerase using TRAP activity assay (Kim et al., Science266:2011, 1997; Weinrich et al., Nature Genetics 17:498, 1997).Expression of hTERT at the mRNA level is evaluated by RT-PCR.

[0127] Cells are telomerized by genetically altering them bytransfection or transduction with a suitable vector, homologousrecombination, or other appropriate technique, so that they express thetelomerase catalytic component (TERT). Particularly suitable is thecatalytic component of human telomerase (hTERT), provided inInternational Patent Publication WO 98/14592. For certain applications,species homologs like mouse TERT (WO 99127113) can also be used.Transfection and expression of telomerase in human cells is described inBodnar et al., Science 279:349, 1998 and Jiang et al., Nat. Genet.21:111, 1999. In another example, hTERT clones (WO 98/14592) are used asa source of hTERT encoding sequence, and spliced into an EcoRI site of aPBBS212 vector under control of the MPSV promoter, or into the EcoRIsite of commercially available pBABE retrovirus vector, under control ofthe LTR promoter. Differentiated or undifferentiated pPS cells aregenetically altered using vector containing supernatants over a 8-16 hperiod, and then exchanged into growth medium for 1-2 days. Geneticallyaltered cells are selected using 0.5-2.5 μg/mL puromycin, andrecultured. They can then be assessed for hTERT expression by RT-PCR,telomerase activity (TRAP assay), immunocytochemical staining for hTERT,or replicative capacity. Continuously replicating colonies will beenriched by further culturing under conditions that supportproliferation, and cells with desirable phenotypes can optionally becloned by limiting dilution.

[0128] In certain embodiments of this invention, pPS cells aredifferentiated, and then genetically altered to express TERT. In otherembodiments of this invention, pPS cells are genetically altered toexpress TERT, and then differentiated. Successful modification toincrease TERT expression can be determined by TRAP assay, or bydetermining whether the replicative capacity of the cells has improved.

[0129] Other methods of immortalizing cells are also contemplated, suchas transforming the cells with DNA encoding the SV40 large T antigen(U.S. Pat. No. 5,869,243, International Patent Publication WO 97/32972).Transfection with oncogenes or oncovirus products is less suitable whenthe cells are to be used for therapeutic purposes. Telomerized cells areof particular interest in applications of this invention where it isadvantageous to have cells that can proliferate and maintain theirkaryotype—for example, in pharmaceutical screening, and in therapeuticprotocols where differentiated or partially differentiated cells areadministered to an individual as part of a protocol to achieve tissueregeneration.

[0130] Use of differentiated cells

[0131] This description provides a method by which large numbers ofpluripotent cells can be produced commercially, and then differentiatedinto committed precursor cells or terminally differentiated cells. Thesecell populations can be used for a number of important research,development, and commercial purposes.

[0132] Preparation of expression libraries and specific antibody

[0133] The differentiated cells of this invention can also be used toprepare specific antibody for phenotypic markers of differentiatedcells. Polyclonal antibodies can be prepared by injecting a vertebrateanimal with cells of this invention in an immunogenic form. Productionof monoclonal antibodies is described in such standard references asHarrow & Lane (1988), U.S. Pat. Nos. 4,491,632, 4,472,500 and 4,444,887,and Methods in Enzymology73B:3 (1981). Other methods of obtainingspecific antibody molecules (optimally in the form of single-chainvariable regions) involve contacting a library of immunocompetent cellsor viral particles with the target antigen, and growing out positivelyselected clones. See Marks et al., New Eng. J. Med. 335:730, 1996,International Patent Publications WO 94/13804, WO 92/01047, WO 90/02809,and McGuiness et al., Nature Biotechnol. 14:1449,1996.

[0134] By positively selecting using pPS of this invention, andnegatively selecting using cells bearing more broadly distributedantigens (such as differentiated embryonic cells) or adult-derived stemcells, the desired specificity can be obtained. The antibodies in turncan be used to identify or rescue cells of a desired phenotype from amixed cell population, for purposes such as costaining duringimmunodiagnosis using tissue samples, and isolating precursor cells fromterminally differentiated cells, and cells of other lineages.

[0135] Differentiated pPS cells of this invention can also be used toprepare mRNA and cDNA libraries that reflect the gene expressionpatterns of these cells. mRNA and cDNA can also be made fromundifferentiated cells, and used to produce subtraction librariesenriched for transcripts that are up- or down-regulated duringdifferentiation. Further information can be found in U.S. patentapplication 09/688,031.

[0136] Screening proliferation factors, differentiation factors, andpharmaceuticals

[0137] pPS cells can be used to screen for factors (such as smallmolecule drugs, peptides, polynucleotides, and the like) or conditions(such as culture conditions or manipulation) that affect thecharacteristics of pPS cells in culture. This system has the advantageof not being complicated by a secondary effect caused by perturbation ofthe feeder cells by the test compound. In one application, growthaffecting substances are tested. The conditioned medium is withdrawnfrom the culture and a simpler medium (such as KO DMEM) is substituted.Different wells are then treated with different cocktails of solublefactors that are candidates for replacing the components of theconditioned medium. Efficacy of each mixture is determined if thetreated cells are maintained and proliferate in a satisfactory manner,optimally as well as in conditioned medium. Potential differentiationfactors or conditions can be tested by treating the cells according tothe test protocol, and then determining whether the treated celldevelops functional or phenotypic characteristics of a differentiatedcell of a particular lineage.

[0138] Feeder-free pPS cultures can also be used for the testing ofpharmaceutical compounds in drug research. Assessment of the activity ofcandidate pharmaceutical compounds generally involves combining thedifferentiated cells of this invention with the candidate compound,determining any resulting change, and then correlating the effect of thecompound with the observed change. The screening may be done, forexample, either because the compound is designed to have apharmacological effect on certain cell types, or because a compounddesigned to have effects elsewhere may have unintended side effects. Twoor more drugs can be tested in combination (by combining with the cellseither simultaneously or sequentially), to detect possible drug-druginteraction effects. In some applications, compounds are screenedinitially for potential toxicity (Castell et al., pp 375-410 in “Invitro Methods in Pharmaceutical Research,” Academic Press, 1997).Cytotoxicity can be determined by the effect on cell viability,survival, and morphology, on the expression or release of certainmarkers, receptors or enzymes, on DNA synthesis or repair. Measured by[³H]-thymidine or BrdU incorporation, or on sister chromatid exchange,determined by metaphase spread. The reader is referred generally to thestandard textbook “In vitro Methods in Pharmaceutical Research”,Academic Press, 1997, and U.S. Pat. No. 5,030,015.

[0139] Genomics

[0140] Suitable methods for comparing expression at the protein levelinclude the immunoassay or immunocytochemistry techniques describedabove. Suitable methods for comparing expression at the level oftranscription include methods of differential display of mRNA (Liang etal., Cancer Res. 52:6966, 1992), and matrix array expression systems(Schena et al., Science 270:467, 1995; Eisen et al., Methods Enzymol.303:179, 1999; Brown et al., Nat. Genet. 21 Suppl 1:33,1999).

[0141] The use of microarray in analyzing gene expression is reviewedgenerally by Fritz et al Science 288:316, 2000; “Microarray BiochipTechnology”, M. Schena ed., Eaton Publishing Company; “Microarrayanalysis”, Gwynne & Page, Science (Aug. 6, 1999 supplement); Pollack etal., Nat Genet 23:41, 1999; Gerhold et al., Trends Biochem. Sci. 24:168,1999; “Gene Chips (DNA Microarrays)”, L. Shi at the Internet URLwww.Gene-Chips.com. Systems and reagents for performing microarrayanalysis are available commercially from companies such as Affymetrix,Inc., Santa Clara Calif.; Gene Logic Inc., Columbia Md.; HySeq Inc.,Sunnyvale Calif.; Molecular Dynamics Inc., Sunnyvale Calif.; Nanogen,San Diego Calif.; and Synteni Inc., Fremont Calif. (acquired by IncyteGenomics, Palo Alto Calif.).

[0142] Solid-phase arrays are manufactured by attaching the probe atspecific sites either by synthesizing the probe at the desired position,or by presynthesizing the probe fragment and then attaching it to thesolid support. A variety of solid supports can be used, includingglasses, plastics, ceramics, metals, gels, membranes, paper, and beadsof various composition. U.S. Pat. No. 5,445,934 discloses a method ofon-chip synthesis, in which a glass slide is derivatized with a chemicalspecies containing a photo-cleavable protecting group. Each site issequentially deprotected by irradiation through a mask, and then reactedwith a DNA monomer containing a photoprotective group. Methods forattaching a presynthesized probe onto a solid support includeadsorption, ultra violet linking, and covalent attachment. In oneexample, the solid support is modified to carry an active group, such ashydroxyl, carboxyl, amine, aldehyde, hydrazine, epoxide, bromoacetyl,maleimide, or thiol groups through which the probe is attached (U.S.Pat. Nos. 5,474,895 and 5,514,785).

[0143] The probing assay is typically conducted by contacting the arrayby a fluid potentially containing the nucleotide sequences of interestunder suitable conditions for hybridization conditions, and thendetermining any hybrid formed. For example, mRNA or DNA in the sample isamplified in the presence of nucleotides attached to a suitable label,such as the fluorescent labels Cy3 or Cy5. Conditions are adjusted sothat hybridization occurs with precise complementary matches or withvarious degrees of homology, as appropriate. The array is then washed,and bound nucleic acid is determined by measuring the presence or amountof label associated with the solid phase. Different samples can becompared between arrays for relative levels of expression, optionallystandardized using genes expressed in most cells of interest, such as aribosomal or housekeeping gene, or as a proportion of totalpolynucleotide in the sample. Alternatively, samples from two or moredifferent sources can be tested simultaneously on the same array, bypreparing the amplified polynucleotide from each source with a differentlabel.

[0144] An exemplary method is conducted using a Genetic Microsystemsarray generator, and an Axon GenePix™ Scanner. Microarrays are preparedby first amplifying cDNA fragments encoding marker sequences to beanalyzed in a 96 or 384 well format. The cDNA is then spotted directlyonto glass slides at a density as high as >5,000 per slide. To comparemRNA preparations from two cells of interest, one preparation isconverted into Cy3-labeled cDNA, while the other is converted intoCy5-labeled cDNA. The two cDNA preparations are hybridizedsimultaneously to the microarray slide, and then washed to eliminatenon-specific binding. Any given spot on the array will bind each of thecDNA products in proportion to abundance of the transcript in the twooriginal mRNA preparations. The slide is then scanned at wavelengthsappropriate for each of the labels, the resulting fluorescence isquantified, and the results are formatted to give an indication of therelative abundance of mRNA for each marker on the array.

[0145] Identifying expression products for use in characterizing andaffecting differentiated cells of this invention involves analyzing theexpression level of RNA, protein, or other gene product in a first celltype, such as a pluripotent precursor cell, or a cell capable ofdifferentiating along a particular pathway; then analyzing theexpression level of the same product in a control cell type; comparingthe relative expression level between the two cell types, (typicallynormalized by total protein or RNA in the sample, or in comparison withanother gene product expected to be expressed at a similar level in bothcell types, such as a house-keeping gene); and then identifying productsof interest based on the comparative expression level.

[0146] Products will typically be of interest if their relativeexpression level is at least about 2-fold, 10-fold, or 100-fold elevated(or suppressed) in differentiated pPS cells of this invention, incomparison with the control. This analysis can optionally becomputer-assisted, by marking the expression level in each cell type onan independent axis, wherein the position of the mark relative to eachaxis is in accordance with the expression level in the respective cell,and then selecting a product of interest based on the position of themark. Alternatively, the difference in expression between the first celland the control cell can be represented on a color spectrum (forexample, where yellow represents equivalent expression levels, redindicates augmented expression and blue represents suppressedexpression). The product of interest can then be selected based on thecolor representing expression of one marker of interest, or based on apattern of colors representing a plurality of markers.

[0147] Genes and proteins that undergo a change in expression levelduring differentiation are of interest for a number of purposes. Forexample, where expression is high in pPS cells and decreases duringdifferentiation can be used as molecular markers of the undifferentiatedstate. Reagents corresponding to these markers, such as antibodies, canbe used, for example, to eliminate undifferentiated pPS cells from apopulation of differentiated cells by immunoaffinity isolation orcomplement-mediated lysis. Where expression is increased duringdifferentiation, the markers can be used in a similar manner to purify,enrich, remove or eliminate specific cell types derived from pPS cells.These markers may serve as indicators of broad classes of celldifferentiation, such as genes or proteins expressed in mesodermal,endodermal or ectodermal lineages, or may serve as markers of highlydifferentiated cell types.

[0148] Genes that are upregulated during expression may also be usefulto influence the differentiation of pPS cells into specific lineages.For instance, the forced expression in undifferentiated pPS cells oftransgenes encoding transcription factors, growth factors, receptors andsignaling molecules can be tested for an ability to influencedifferentiation into specific cell lineages.

[0149] Once the sequence of mRNA preferentially expressed or repressedin differentiated cells is determined, it can be used in the manufactureof polynucleotides that contain such sequences, polypeptides theyencode, and antibody specific for the polypeptides. Oligonucleotides ofless than ˜50 base pairs are conveniently prepared by chemicalsynthesis, either through a commercial service or by a known syntheticmethod, such as solid phase synthesis (Hirose et al., Tetra. Lett.19:2449-2452, 1978; U.S. Pat. No. 4,415,732). Polynucleotides can alsobe manufactured by PCR amplification using a template with the desiredsequence (U.S. Pat. Nos. 4,683,195 and 4,683,202). Production scaleamounts of large polynucleotides are conveniently obtained by insertingthe desired sequence into a suitable cloning vector, and eitherreproducing the clone, or transfecting the sequence into a suitable hostcell. Short polypeptides can be prepared by solid-phase chemicalsynthesis: see Dugas & Penney, Bioorganic Chemistry, Springer-Verlag NYpp 54-92 (1981). Longer polypeptides are conveniently manufactured bytranslation in an in vitro translation system, or by expression in asuitable host cell (U.S. Pat. No. 5,552,524). Polyclonal and monoclonalantibody specific for polypeptides encoded by mRNA and cDNA of thisinvention can be obtained by determining amino acid sequence from aprotein encoding region in an expression library, and immunizing ananimal or contacting an immunocompetent cell or particle with a proteincontaining the determined sequence, according to standard techniques.

[0150] Therapeutic Compositions

[0151] Differentiated cells of this invention can also be used fortissue reconstitution or regeneration in a human patient in needthereof. The cells are administered in a manner that permits them tograft to the intended tissue site and reconstitute or regenerate thefunctionally deficient area.

[0152] In one example, neural stem cells are transplanted directly intoparenchymal or intrathecal sites of the central nervous system,according to the disease being treated. Grafts are done using singlecell suspension or small aggregates at a density of 25,000-500,000 cellsper μL (U.S. Pat. No. 5,968,829). The efficacy of neural celltransplants can be assessed in a rat model for acutely injured spinalcord as described by McDonald et al. (Nat. Med. 5:1410, 1999. Asuccessful transplant will show transplant-derived cells present in thelesion 2-5 weeks later, differentiated into astrocytes,oligodendrocytes, and/or neurons, and migrating along the cord from thelesioned end, and an improvement in gate, coordination, andweight-bearing.

[0153] The efficacy of cardiomyocytes can be assessed in an animal modelfor cardiac cryoinjury, which causes 55% of the left ventricular walltissue to become scar tissue without treatment (Li et al., Ann. Thorac.Surg. 62:654, 1996; Sakai et al., Ann. Thorac. Surg. 8:2074, 1999, Sakaiet al., J. Thorac. Cardiovasc. Surg. 118:715, 1999). Successfultreatment will reduce the area of the scar, limit scar expansion, andimprove heart function as determined by systolic, diastolic, anddeveloped pressure. Cardiac injury can also be modeled using anembolization coil in the distal portion of the left anterior descendingartery (Watanabe et al., Cell Transplant. 7:239, 1998), and efficacy oftreatment can be evaluated by histology and cardiac function.Cardiomyocyte preparations embodied in this invention can be used intherapy to regenerate cardiac muscle and treat insufficient cardiacfunction (U.S. Pat. No. 5,919,449 and WO 99/03973).

[0154] Hepatocytes and hepatocyte precursors can be assessed in animalmodels for ability to repair liver damage. One such example is damagecaused by intraperitoneal injection of D-galactosamine (Dabeva et al.,Am. J. Pathol. 143:1606, 1993). Efficacy of treatment can be determinedby immunocytochemical staining for liver cell markers, microscopicdetermination of whether canalicular structures form in growing tissue,and the ability of the treatment to restore synthesis of liver-specificproteins. Liver cells can be used in therapy by direct administration,or as part of a bioassist device that provides temporary liver functionwhile the subject's liver tissue regenerates itself following fulminanthepatic failure.

[0155] Cells prepared according to this invention that are useful forhuman or veterinary therapy are optimally supplied in a pharmaceuticalcomposition, comprising an isotonic excipient prepared undersufficiently sterile conditions for human administration. For generalprinciples in medicinal formulation, the reader is referred to CellTherapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge UniversityPress, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister& P. Law, Churchill Livingstone, 2000. The compositions may be packagedwith written instructions for use of the cells in tissue regeneration,or restoring a therapeutically important metabolic function.

[0156] The examples that follow are provided by way of furtherillustration, and are not meant to imply any limitation in the practiceof the claimed invention.

EXAMPLES Example 1

[0157] Feeder-Free Passage of hES Cells

[0158] Undifferentiated hES cells isolated on primary mouse embryonicfeeder cells were propagated in the absence of feeder cells. The culturewells were coated with Matrigel®, and the cells were cultured in thepresence of conditioned nutrient medium obtained from a culture ofirradiated primary fibroblasts.

[0159] Preparation of conditioned media (CM) from primary mouseembryonic fibroblasts (mEF):

[0160] Fibroblasts were harvested from T150 flasks by washing once withCa⁺ /Mg⁺⁺ free PBS and incubating in 1.5-2 mL trypsin/EDTA (Gibco) forabout 5 min. After the fibroblasts detached from the flask, they werecollected in mEF media (DMEM+10% FBS). The cells were irradiated at 4000rad (508 sec at 140 kV: shelf setting 6 in a Torrex generator), countedand seeded at about 55,000 cells cm⁻² in mEF media (525,000 cells/wellof a 6 well plate). After at least 4 hours the media were exchanged withSR containing ES media, using 3-4 mL per 9.6 cm well of a 6 well plate.Conditioned media was collected daily for feeding of hES cultures.Alternatively, medium was prepared using mEF plated in culture flasks,exchanging medium daily at 0.3-0.4 mL cm⁻². Before addition to the hEScultures, the conditioned medium was supplemented with 4 ng/mL of humanbFGF (Gibco). Fibroblast cultures were used in this system for about 1week, before replacing with newly prepared cells.

[0161] Matrigel® coating:

[0162] Growth Factor Reduced Matrigel® or regular Matrigel®(Becton-Dickinson, Bedford Mass.) was thawed at 4° C. The Matrigel® wasdiluted 1:10 to 1:500 (typically 1:30) in cold KO DMEM. 0.75-1.0 mL ofsolution was added to each 9.6 cm² well, and incubated at roomtemperature for 1 h, or at 4° C. at least overnight. The coated wellswere washed once with cold KO DMEM before adding cells. Plates were usedwithin 2 h after coating, or stored in DMEM at 4° C. and used within ˜1week.

[0163] Human ES culture:

[0164] Undifferentiated hES colonies were harvested from hES cultures onfeeders as follows. Cultures were incubated in ˜200 U/mL collagenase IVfor about 5 minutes at 37° C. Colonies were harvested by pickingindividual colonies up with a 20 μL pipet tip under a microscope or byscraping and dissociating into small clusters in conditioned medium(CM). These cells were then seeded onto Matrigel® in conditioned mediaat 15 colonies to each 9.6 cm² well (if 1 colony is ˜10,000 cells, thenthe plating density is ˜15,000 cells cm⁻²).

[0165] The day after seeding on Matrigel®, hES cells were visible assmall colonies (˜100-2,000 cells) and there were cells in between thecolonies that appeared to be differentiating or dying. As the hES cellsproliferated, the colonies became quite large and very compact,representing the majority of surface area of the culture dish. The hEScells in the colonies had a high nucleus to cytoplasm ratio and hadprominent nucleoli, similar to hES cells maintained on feeder cells. Atconfluence, the differentiated cells in between the colonies representedless than 10% of the cells in the culture.

[0166] Six days after seeding, the cultures had become almost confluent.The cultures were split by incubating with 1 mL ˜200 U/mL Collagenase IVsolution in KO DMEM for ˜5 minutes at 37° C. The collagenase solutionwas aspirated, 2 mL hES medium was added per well, and the hES cellswere scraped from the dish with a pipette. The cell suspension wastransferred to a 15 mL conical tube, brought up to a volume of 6 mL, andgently triturated to dissociate the cells into small clusters of 10-2000cells. The cells were then re-seeded on Matrigel® coated plates in CM,as above. Cells were seeded at a 1:3 or 1:6 ratio, approximately 90,000to 170,000 cells cm⁻², making up the volume in each well to 3 mL. Mediumwas changed daily, and the cells were split and passaged again at 13 dand again at 19 d after initial seeding.

[0167] Undifferentiated hES cells express SSEA-4, Tra-1-60, Tra-1-81,OCT-4, and hTERT. In order to assess whether the cells maintained infeeder-free conditions retained these markers, cells were evaluated byimmunostaining, reverse transcriptase PCR amplification, and assay fortelomerase activity. As assayed by fluorescence-activated cell sorting,cells on Matrigel®, laminin, fibronectin or collagen IV expressedSSEA-4, Tra-1-60 and Tra-1-81. There was very little expression ofSSEA-1, a glycolipid that is not expressed by undifferentiated hEScells. Immunocytochemistry analysis shows that SSEA-4, Tra-1-60,Tra-1-81, and alkaline phosphatase were expressed by the hES colonies onMatrigel®D or laminin, as seen for the cells on feeders—but not by thedifferentiated cells in between the colonies.

[0168]FIG. 1 shows OCT-1 and hTERT expression of Hi cells on feeders andoff feeders, as detected by reverse-transcriptase PCR amplification. Forradioactive relative quantification of individual gene products,QuantumRNA™ Alternatel8S Internal Standard primers (Ambion, Austin Tex.,USA) were employed according to the manufacturer's instructions.Briefly, the linear range of amplification of a particular primer pairwas determined, then coamplified with the appropriate mixture ofalternate18S primers:competimers to yield PCR products with coincidinglinear ranges. Before addition of AmpliTaq™ (Roche) to PCR reactions,the enzyme was pre-incubated with the TaqStart™ antibody (ProMega)according to manufacturer's instructions. Radioactive PCR reactions wereanalyzed on 5% non-denaturing polyacrylamide gels, dried, and exposed tophosphoimage screens (Molecular Dynamics) for 1 hour. Screens werescanned with a Molecular Dynamics Storm 860 and band intensities werequantified using ImageQuant™ software. Results are expressed as theratio of radioactivity incorporated into the hTERT or OCT-4 band,standardized to the radioactivity incorporated into the 18s band.

[0169] Primers and amplification conditions for particular markers areas follows. OCT-4: Sense (SEQ. ID NO:1) 5′-CTTGCTGCAG AAGTGGGTGGAGGAA-3′; Antisense (SEQ. ID NO:2) 5′-CTGCAGTGTG GGTTTCGGGC A-3′;alternate18:competimers 1:4; 19 cycles (94° 30 sec; 60° 30 sec; 72° 30sec). hTERT: Sense (SEQ. ID NO:3) 5′-CGGMGAGTG TCTGGAGCAA-3′; Antisense(SEQ. ID NO:4) 5′-GGATGAAGCG GAGTCTGGA-3′; alternate 18:competimers1:12; 34 cycles (94° 30 sec; 60° 30 sec; 72° 30 sec).

[0170] The transcription factor OCT-4 is normally expressed in theundifferentiated hES cells and is down-regulated upon differentiation.The cells maintained on Matrigel® or laminin in conditioned medium (CM)for 21 days express OCT-4, whereas cells maintained in Matrigel® inunconditioned regular medium (RM) did not. Cells maintained onfibronectin or collagen IV, which showed a large degree ofdifferentiation, expressed lower levels of OCT-4 compared to cells onfeeders, Matrigel® or laminin.

[0171] Telomerase activity was measured by TRAP assay (Kim et al.,Science 266:2011, 1997; Weinrich et al., Nature Genetics 17:498, 1997).All the cultures conditions showed positive telomerase activity after 40days on Matrigel®, laminin, fibronectin or collagen IV in mEFconditioned medium.

Example 2

[0172] Direct Differentiation of hES cells

[0173] Differentiation using standard methods of aggregate formation wascompared with a technique of this invention in which cells aredifferentiated by plating directly onto a solid surface under certainconditions.

[0174] For the aggregate differentiation technique, monolayer culturesof rhesus and human ES lines were harvested by incubating in CollagenaseIV for 5-20 min, and the cells were scraped from the plate. The cellswere then dissociated and plated in non-adherent cell culture plates inFBS-containing medium (20% non-heat-inactivated FBS (Hyclone),supplemented with 0.1 mM non-essential amino acids, 1 mM glutamine, 0.1mM β-mercaptoethanol. The EBs were fed every other day by the additionof 2 mL of medium per well (6 well plate). When the volume of mediumexceeded 4 mL/well, the EBs were collected and resuspended in freshmedium. The plates were placed into a 37° C. incubator, and in someinstances, a rocker was used to facilitate maintaining aggregates insuspension. After 4-8 days in suspension, aggregate bodies formed andwere plated onto a substrate to allow for further differentiation.

[0175] For the direct differentiation technique, suspensions of rhesusand human ES cells were prepared in a similar fashion. The cells werethen dissociated by trituration to clusters of ˜50-100 cells, and platedonto glass coverslips treated with poly-ornithine. The cells weremaintained in serum containing medium, or defined medium for 7-10 daysbefore analysis. Cells were tested by immunoreactivity for β-tubulin IIIand MAP-2, which are characteristic of neurons, and glial fibrillaryacidic protein (GFAP), which is characteristic of astrocytes.

[0176] Six different ES lines differentiated into cells bearing markersfor neurons and astrocytes, using either the aggregate or directdifferentiation technique. In cultures derived from rhesus ES cells,percentage of aggregates that contained neurons ranged from 49% to 93%.In cultures derived from human ES cells, the percentage of aggregatescontaining neurons ranged from 60% to 80%. Double labeling for GABA andβ-tubulin indicated that a sub-population of the neurons express theinhibitory neurotransmitter GABA. Astrocytes and oligodendrocytes wereidentified with GFAP immune reactivity and GaIC immune reactivity,respectively. Therefore, the human and rhesus ES cells have the capacityto form all three major cell phenotypes in the central nervous system.

[0177] The effect of several members of the neurotrophin growth factorfamily was examined. hES cells were differentiated by harvesting withcollagenase, dissociating, and reseeding onto poly-ornithine coatedcover slips. The cells were plated into DMEM/F12 +N2+10% FBS overnight.The following day, the serum was removed from the medium and replacedwith 10 ng/mL human bFGF and the growth factor being tested. After 24hours, bFGF was removed from the medium. These cultures were fed everyother day. They were fixed after 7 days of differentiation andimmunostained for analysis. The number of neurons was evaluated bycounting cells positive for β-tubulin. Cultures maintained in thepresence of 10 ng/mL brain derived neurotrophic factor (BDNF) formedapproximately 3-fold more neurons than the control cultures. Culturesmaintained in neurotrophin-3 (1 ng/mL) formed approximately 2-fold moreneurons than control cultures.

[0178] To assess cardiomyocyte formation, EBs were transferred togelatin-coated plates or chamber slides after 4 days in the suspensioncultures. The EBs attached to the surface after seeding, proliferatedand differentiated into different types of cells. Spontaneouslycontracting cells were observed in various regions of the culture atdifferentiation day 8 and the number of beating regions increased untilabout day 10. In some cases, more than 75% of the EBs had contractingregions. Beating cells were morphologically similar to mouse EScell-derived beating cardiomyocytes. In these cultures 100% of thecontracting areas were immunoreactive with cardiac troponin I (cTnI),while minimal immunoreactivity was observed in the non-beating cells.

[0179] Cultures of differentiated EBs were subjected to Western blotanalysis using monoclonal antibody against cTnl. This assay gave astrong 31 kDa protein signal, corresponding to the size of the purifiednative human cTnI. It was detected in differentiated human ES cellscontaining contracting cells, but not in undifferentiated ES cells ordifferentiated cultures with no evidence of contracting cells. As acontrol, the blot was reprobed with β-actin specific antibody,confirming the presence of similar amounts of proteins in all samples.

[0180] In other experiments, EBs were cultured for 8 or 16 days andmaintained as adherent cultures for an additional 10 days. RNA wasprepared from the differentiated human ES cells and semiquantitativeRT-PCR was performed to detect the relative expression of theendoderm-specific products (α-anti-trypsin, AFP, and albumin. Low levelsof (α₁-anti-trypsin and AFP were detected in the undifferentiatedcultures; little or no albumin was detected in the same cultures. All 3markers were detected at significantly higher levels afterdifferentiation. Expression of all 3 endoderm markers was higher incultures derived from 8 day embryoid bodies than 16 day embryoid bodies.

Example 3

[0181] Direct Differentiation of hES to Hepatocyte-Like Cells withoutForming Embryoid Bodies

[0182] This experiment demonstrated the use of the directdifferentiation technique for deriving human ES cells into a relativelyuniform population of cells with phenotypic markers of hepatocytes.

[0183] The hES cells were maintained in undifferentiated cultureconditions (Matrigel® plus mEF conditioned medium) for 2-3 days aftersplitting. At this time, the cells were 50-60% confluent and the mediumwas exchanged with unconditioned SR medium containing 1% DMSO.

[0184] The cultures were fed daily with SR medium for 4 days and thenexchanged into unconditioned SR medium containing 2.5% Na-butyrate(which was previously identified as a hepatocyte differentiation agent).The cultures were fed daily with this medium for 6 days; at which timeone half of the cultures were evaluated by immunocytochemistry. Theother half of the cultures were harvested with trypsin and replated ontocollagen, to further promote enrichment for hepatocyte lineage cells.Immunocytochemistry was then performed on the following day.

[0185] As shown in Table 1, the cells which underwent the finalre-plating had -5-fold higher albumin expression, similar α₁-antitrypsinexpression and 2-fold less cytokeratin expression than the cells notre-plated. The secondary plating for the cells is believed to enrich forthe hepatocyte-like cells. TABLE 1 Phenotype of Differentiated Cells Notrypsinization Trypsinization Antibody Specificity % positive % positive(no primary antibody) 0 0 (IgG1 control) 0 0 albumin  11% 63%α₁-antitrypsin >80% >80%  α-fetoprotein 0 0 Cytokeratin 8 >80% 45%Cytokeratin 18 >80% 30% Cytokeratin 19 >80% 30% glycogen 0 >50% 

[0186] Adjustments to culture conditions are shown in Table 2.Hepatocyte Culture Medium is purchased from Clonetics; Strom's Medium isprepared as described in Runge et al., Biochem. Biophys. Res. Commun.265:376, 1999. The cell populations obtained are assessed byimmunocytochemistry and enzyme activity. TABLE 2 Direct DifferentiationProtocols Further differentiation Undifferentiated cellsPre-differentiation Hepatocyte induction (Groups 1-3 only; (untilconfluent) (4 days) (6 days) 4 days) Feeder-free conditions 20% SRmedium + 20% SR medium + HCM + 30 ng/mL hEGF + 1% DMSO 1% DMSO + 10ng/mL TGF-α + 2.5 mM butyrate 30 ng/mL HGF + 1% DMSO + 2.5 mM butyrateFeeder-free conditions 20% SR medium + 20% SR medium + 20% SR medium +1% DMSO 1% DMSO + 30 ng/mL hEGF + 2.5 mM butyrate 10 ng/mL TGF-α + 30ng/mL HGF + 1% DMSO + 2.5 mM butyrate Feeder-free conditions 20% SRmedium + 20% SR medium + Strom's medium + 1% DMSO 1% DMSO + 30 ng/mLhEGF + 2.5 mM butyrate 10 ng/mL TGF-α + 30 ng/mL HGF + 1% DMSO + 2.5 mMbutyrate Feeder-free conditions 20% SR medium + HCM + 30 ng/mL hEGF + 1%DMSO 10 ng/mL TGF-α + 30 ng/mL HGF + 1% DMSO + 2.5 mM butyrateFeeder-free conditions 20% SR medium + 20% SR medium + 1% DMSO 30 ng/mLhEGF + 10 ng/mL TGF-α + 30 ng/mL HGF + 1% DMSO + 2.5 mM butyrateFeeder-free conditions 20% SR medium + Strom's medium + 1% DMSO 30 ng/mLhEGF + 10 ng/mL TGF-α + 30 ng/mL HGF + 1% DMSO + 2.5 mM butyrateFeeder-free conditions HCM + 30 ng/mL hEGF + HCM + 30 ng/mL hEGF + 10ng/mL TGF-α + 10 ng/mL TGF-α + 30 ng/mL HGF + 30 ng/mL HGF + 1% DMSO 1%DMSO + 2.5 mM butyrate Feeder-free conditions 20% SR medium + 20% SRmedium + 30 ng/mL hEGF + 30 ng/mL hEGF + 10 ng/mL TGF-α + 10 ng/mLTGF-α + 30 ng/mL HGF + 30 ng/mL HGF + 1% DMSO 1% DMSO + 2.5 mM butyrateFeeder-free conditions Strom's medium + Strom's medium + 30 ng/mL hEGF +30 ng/mL hEGF + 10 ng/mL TGF-α + 10 ng/mL TGF-α + 30 ng/mL HGF + 30ng/mL HGF + 1% DMSO 1% DMSO + 2.5 mM butyrate

[0187] Other additives tested in the subsequent (4-day) maturation stepinclude factors such as FGF-4, and oncostatin M in the presence ofdexamethazone.

[0188]FIG. 2 shows the effect of HCM on maturation of hES-derived cells.Left column: 10 X magnification; Right column: 40 X magnification. By 4days in the presence of butyrate, more than 80% of cells in the cultureare large in diameter, containing large nuclei and granular cytoplasm(Row A). After 5 days in SR medium, the cells were switched to HCM. Twodays later, many cells are multinucleated, and have a large polygonalshape (Row B). By 4 days in HCM, multinucleated polygonal cells arecommon, and have a darker cytosol (Row C), by which criteria theyresemble freshly isolated human adult hepatocytes (Row D) or fetalhepatocytes (Row E).

Example 4

[0189] Microarray Analysis of Expression by Undifferentiated andDifferentiated Cells

[0190] An analysis of differential gene expression was performed bycontrasting mRNA from undifferentiated H9 cultures with mRNA fromcorresponding EBs. The EBs were maintained in growth medium for 8 days,or kept in growth medium for 4 days, followed by 4 days of treatmentwith 0.5 μM retinoic acid. EBs were harvested after 2 d, 4 d, or 8 d andthe resulting mRNA was compared directly with mRNA from undifferentiatedcultures. This analysis tracks the transformation of a relativelyhomogenous cell population into a complex mix of differentiated celltypes, and thus the readouts are affected both by the magnitude of thechange in gene expression, and, in the case of expression changesspecific to a differentiated cell type, by the representation of thatcell type in the culture. The arrays used in these experiments sampleapproximately 10,000 cDNAs selected to represent a large portion ofcharacterized human genes.

[0191] Total RNA was harvested from human ES cultures or theirdifferentiated derivatives using the Qiagen RNAeaSy™ Miniprep kitaccording to the manufacturer's instructions. RNA was quantified bymeasuring ultraviolet absorption at 260 nm. Poly A⁺ mRNA was preparedfrom the total RNA preparations using Qiagen Oligotex™ Miniprepsaccording to the instructions of the manufacturer. Final mRNApreparations were quantified by A₂₆₀ measurements, then visuallyinspected following electrophoresis on native agarose gels. Sample RNAswere sent to a contract laboratory (Incyte Pharmaceuticals, Palo Alto,Calif.) for conversion into Cy3- or Cy5 labeled cDNA probe, which wassubsequently hybridized to UNIGEM™ 1.0 arrays.

[0192] Following processing of the hybridized arrays, fluorescencemeasurements were quantified and the results returned for analysis.Probe pairings were performed with samples from undifferentiated EScells in the Cy3 channel, and the differentiated ES cell samples in theCy5 channel. A change in expression (as measured by comparing the Cy3and Cy5 channels) was generally considered significant if the differencewas at least 2.5-fold.

[0193]FIG. 3 shows the expression analysis of embryoid body (EB) cells.The numbers in the matrix compare expression at the mRNA level withexpression in the undifferentiated hES cell line from which the EBs werederived. Numbers 1.1 and above represent a proportional increase inexpression in EB cells; numbers −1.1 and below represent a proportionaldecrease in expression. The four columns show results obtained from EBsin standard suspension culture for 2, 4, or 8 days; or cultured 4 daysin regular medium and 4 days in medium containing retinoic acid (4 d−/4d+).

[0194] The differentiation of hES cells involves the activation andrepression of many genes, including ESTs with no known function.Interestingly, the addition of retinoic acid to the suspension culturefor the final 4 days of differentiation had relatively minor effect onthe gene expression pattern (compare 4 d−/4 d+ with 8 d).

[0195] Genes whose expression is reduced during differentiation sample awide range of functions, including metallothioneins, growth factors(e.g., FGF9), secreted cysteine-rich proteins (e.g., osteopontin,AGF-BP5, Cyr61, connective tissue growth factor), the selenium donorprotein selD, and many others. In general, the most significantalterations in expression occur after 4 days of suspension culture, andcorrespond with the onset of changes in cell morphologies. Of interest,the expression of two genes involved in the catabolism of α-D-Glucosephosphate, UDP-glucose phosphorylase and phosphoglucomutase, aredramatically reduced upon differentiation, suggesting a potentialalteration in glucose metabolism.

[0196] The arrays used in these experiments do not contain cDNA featurescorresponding to hTERT; however, a marked decrease in the expression ofthe mRNA for TRF1 was observed. TRF1 is a principal telomere bindingfactor whose expression has been correlated with a shortening oftelomere lengths. Thus, the expression of both positive (hTERT) andnegative (TRF1) regulators of telomere length is reduced during ES celldifferentiation.

[0197] Several genes associated with visceral endoderm and early hepaticdifferentiation were predominant in this analysis, includingα-fetoprotein, apoplipoprotein A-II, apoplipoprotein AI regulatoryprotein-1, α₁-antitrypsin, and the α, β, and γ chains of fibrinogen.This induction is apparent within 2 days of differentiation, and is notsubstantially affected by retinoid treatment. The induced expression ofcellular retinoic acid binding proteins 1 and 2 (CRABP I, II) is notobserved in retinoid treated cultures, consistent with a proposednegative feedback loop in which retinoids specifically inhibit thetranscription of the promoter of the CRAB I gene.

[0198] Expression of the IL-6 receptor gp130 is low in hES cultures, andis induced upon differentiation. These results provide a molecular basisfor the lack of LIF responsiveness in hES cultures (Thomson et al.,1999; Reubinoff et al., 2000) and indicate a substantially differentrole for gp130 in human vs. mouse ES cells, where LIF signaling isdirectly implied in the maintenance of the undifferentiated state.

[0199] Other differentiation-induced genes include the protein homologspleiotropin and midkine. These secreted cytokine have proposed roles asmitogens for neuronal and hepatic cell types, or as generalizedangiogenic factors (Owada et al., 1999; Sato et al., 1999), and as suchmay play a similar role in ES cell differentiation. The induction of DNAbinding proteins, such as homeobox b5 protein and meis1, likely reflectsthe central role of transcriptional regulators in differentiationprocesses.

Example 5

[0200] Direct Differentiation of hES Cells to Neurons

[0201] This study evaluated various paradigms for differentiating humanES cells into neurons without the formation of embryoid bodies.

[0202] A strategy was developed in which the test factors were placedinto groups based on homology and/or functional redundancy (Table 3).Grouping factors increases the likelihood that an activity associatedwithin that group will be elicited on the ES cell population. Thehypothesis is that certain factors within the mixture will initiate adifferentiation cascade. As differentiation proceeds, and the receptorexpression profile of the cells change, they will become responsive toother factors in the mixture.

[0203] Providing a complex mixture of factors continuously over thetreatment period avoids the need to define exactly how and when theresponsiveness of the cells changes. When a mixture is identified thatelicits the desired differentiation process, it can be systematicallysimplified to achieve a minimal optimal mixture. After further testing,minimal treatment may ultimately comprise one, two, three, or more ofthe factors listed, used either simultaneously or in sequence accordingto the empirically determined protocol. TABLE 3 Test Factor Groups Group1 Group 2 Group 3 Neurotrophins Mitogens Stem Cell Factors 30 ng/mL NGF30 ng/mL EGF 8 ng/mL LIF 30 ng/mL NT-3 30 ng/mL FGF-2 3 ng/mL IL-6 30ng/mL NT-4 37 ng/mL FGF-8b 3 ng/mL IL-11 30 ng/mL BDNF 30 ng/mL IGF-l 3ng/mL SOF 30 ng/mL PDGF-AA 30 ng/mL CNTF Group 4 Differentiation FactorsGroup 5 Group 6 TGF-f3 Superfamily TGF-β Superfamily AntagonistsDifferentiation Factor 150 ng/mL Noggin 37 ng/mL SHH 30 ng/mL BMP-2 30ng/mL Follistatin 37 ng/mL GDF-5 3 ng/mL GDNF 30 ng/mL Neurturin Group 7Group 8 Group 9 Neurotrophic Factor Differentiation Factor SurvivalFactor/Antioxidant 37 ng/mL Midkine 17 μM Retinoic Acid 166 μM AscorbicAcid Group 10 Differentiation Factor/ Group 11 Neurotransmitter SurvivalFactor 10 gM Dopamine 100 μM Dibutyryl cAMP

[0204] The experiment was conducted as follows. Monolayer cultures of ahuman ES cell line were harvested by incubating in Collagenase IV for5-10 min, and then scraping the cells from the plate. The cells weredissociated by trituration and plated at subconfluence onto 96 welltissue culture plates pretreated with growth factor-reduced Matrigel® inKnockout DMEM medium (Gibco BRL) with Knockout Serum Replacement (GibcoBRL) conditioned 24 h by mouse embryonic fibroblasts. One day afterplating, the medium was replaced with Neurobasal Medium (Gibco BRL)supplemented with 1 mM glutamine, B27 supplement (Gibco BRL) and groupsof test factors as described below. The cells were fed daily with freshNeurobasal Medium containing glutamine, B27, and test factors for 11days.

[0205] After 11 days, the cells were harvested by incubation in trypsinfor 5-10 min, replated at a 1:6 dilution onto 96 well tissue cultureplates pretreated with laminin, and fed daily with fresh NeurobasalMedium containing glutamine, B27 and test factors for an additional 5days. Cells were fixed for 20 min in 4% paraformaldehyde, and stainedwith antibodies to the early neuronal marker, β-Tubulin-III, the lateneuronal marker, MAP-2, and tyrosine hydroxylase, an enzyme associatedwith dopaminergic neurons. Cell nuclei were labeled with DAPI, andquantified by visual inspection. Results are shown in Table 4. TABLE 4Direct Differentiation of hES Cells to Neurons βTubulin-III TyrosineHydroxylase positive MAP-2 positive Test Compound Groups Cells/ positiveCells/ Included in Cell Culture Well % Total Cells/Well Well % TotalControl 102 — 2 1 — Treatment A: 1, 2, 3, 4, 6, 7, 8, 9, 10, 11  0 0 0 0— Treatment B: 1, 2, 3, 5, 6, 7, 8, 9, 10, 11 362  6% 132  14  0.2%Treatment C: 1, 2, 4, 6, 7, 8, 9, 10, 11 — — — — — Treatment D: 1, 2, 5,6, 7, 8, 9, 10, 11 378 11% 162  16  0.5% Treatment E: 1, 3, 4, 6, 7, 8,9, 10, 11  6 — 2 4 — Treatment F: 1, 3, 5, 6, 7, 8, 9, 10, 11 282 12%92  4 0.2% Treatment G: 1, 4, 6, 7, 8, 9, 10, 11  17 — 0 2 —

[0206] In another experiment, cells were cultured in Neurobasal mediumsupplemented with glutamine, B27 and groups of test factors as before,harvested with trypsin at 8 days, and replated for 5 days. Results areshown in Table 5. TABLE 5 Direct Differentiation of hES Cells to NeuronsPercent of Tyrosine MAP-2 βTubulin-III MAP-2 Hydroxylase positive cellsTest Compound Groups positive positive positive also positive Includedin Cell Culture Cells/Well Cells/Well Cells/Well for TH Control  4 4 0Treatment A: 1, 2, 3, 4, 6, 7, 8, 9, 10, 11  12 8 3 Treatment B: 1, 2,3, 5, 6, 7, 8, 9, 10, 11 268 12  4 Treatment C: 1, 2, 4, 6, 7, 8, 9, 10,11  12 0 0 Treatment D: 1, 2, 5, 6, 7, 8, 9, 10, 11 372 48  7 15%Treatment E: 1, 3, 4, 6, 7, 8, 9, 10, 11  0 0 0 Treatment F: 1, 3, 5, 6,7, 8, 9, 10, 11 196 56  0 Treatment G: 1, 4, 6, 7, 8, 9, 10, 11  16 0 9

[0207] Several treatment paradigms induced the direct differentiation ofneurons. Treatments that included Group 5 factors (noggin andfollistatin) were the most effective.

[0208]FIG. 4 shows exemplary fields of differentiated cells obtainedusing Treatment B, Treatment D, and Treatment F, and stained forβ-tubulin-III. About 5-12% of the cells are neurons, based on morphologyand β-tubulin-III staining. About ⅓ of these are mature neurons, basedon MAP-2 staining. About 2-5% of total neurons (5-15% of MAP-2 positiveneurons) also stained for tyrosine hydroxylase, which is consistent witha dopaminergic phenotype.

[0209] It will be recognized that the compositions and proceduresprovided in the description can be effectively modified by those skilledin the art without departing from the spirit of the invention embodiedin the claims that follow.

1 4 1 25 DNA Artificial Sequence primer 1 cttgctgcag aagtgggtgg aggaa 252 21 DNA Artificial Sequence primer 2 ctgcagtgtg ggtttcgggc a 21 3 20DNA Artificial Sequence primer 3 cggaagagtg tctggagcaa 20 4 19 DNAArtificial Sequence primer 4 ggatgaagcg gagtctgga 19

What is claimed as the invention is:
 1. A method for producing differentiated cells from a donor culture of undifferentiated primate pluripotent stem (pPS) cells, comprising: a) preparing a suspension of pPS cells from the undifferentiated donor culture; b) replating and culturing the suspended cells on a solid surface so that they differentiate without forming embryoid bodies; and c) harvesting differentiated cells from the solid surface.
 2. A method for obtaining differentiated cells from a donor culture of undifferentiated primate pluripotent stem (pPS) cells, comprising: a) culturing the pPS cells on a solid surface in an environment essentially free of feeder cells; b) changing medium used to culture the cells so that they differentiate before there is overgrowth or formation of colonies; and c) harvesting differentiated cells from the solid surface.
 3. The method of claim 1, wherein the donor culture comprises both undifferentiated pPS cells and feeder cells, which are replated onto a solid surface without adding fresh feeder cells.
 4. The method of claim 1, wherein the donor culture is essentially free of feeder cells, which are replated on a solid surface without any extracellular matrix.
 5. The method of claim 1, wherein the solid surface bears a polycation.
 6. The method of claim 5, wherein the polycation is polyomithine.
 7. The method of claim 1, wherein the cells are cultured after replating in a medium containing a factor that promotes differentiation.
 8. The method of claim 7, wherein the factor is Brain Derived Neurotrophic Factor (BDNF) or Neutrotrophin-3 (NT-3).
 9. The method of claim 2, wherein the changed medium is essentially free of fibroblast growth factor.
 10. The method of claim 2, wherein the changed medium contains Brain Derived Neurotrophic Factor (BDNF) or Neutrotrophin-3 (NT-3).
 11. The method of claim 2, wherein the changed medium contains noggin or follistatin.
 12. The method of claim 1, whereby cells cultured on the solid surface differentiate to precursor cells committed to a restricted cell lineage and capable of proliferation.
 13. The method of claim 12, wherein the precursor cells are ectodermal cells.
 14. The method of claim 13, wherein the precursor cells are committed to the neuroectoderm lineage.
 15. The method of claim 14, wherein the precursor cells are cells of the mesoderm, endoderm or visceral endoderm.
 16. The method of claim 1, whereby cells cultured on the solid surface become fully differentiated cells.
 17. The method of claim 16, wherein the fully differentiated cells are neurons or glial cells.
 18. The method of claim 17, wherein at least ˜10% of the cells staining positive for MAP-2 are also positive for tyrosine hydroxylase.
 19. Committed precursor cells prepared according to the method of claim
 12. 20. Fully differentiated cells prepared according to the method of claim
 16. 21. A method of screening a compound for cellular toxicity or modulation, comprising combining the compound with a committed or differentiated cell prepared according to the method of claim 1, determining any phenotypic or metabolic changes in the cell that result from contact with the compound, and correlating the change with cellular toxicity or modulation.
 22. A method for obtaining a polynucleotide comprising a nucleotide sequence contained in an mRNA that is expressed at a different level in committed or differentiated cells prepared according to the method of claim 1, compared with undifferentiated primate pluripotent stem (pPS) cells, the method comprising: a) determining the level of expression of a plurality of mRNAs in committed or differentiated cells, in comparison to the level of expression of the same mRNAs in undifferentiated pPS cells; b) identifying an mRNA expressed at a different level in the committed or differentiated cells, relative to the undifferentiated pPS cells; and c) preparing a polynucleotide comprising a nucleotide sequence of at least 30 consecutive nucleotides contained in the identified mRNA. 